Full text of DOI:10.1016/S0014-2999(00)00126-6

European Journal of Pharmacology 394 Ž2000. 139–148
www.elsevier.nlrlocaterejphar
Acute neurogenic airway plasma exudation and edema induced by
inhaled wood smoke in guinea pigs: role of tachykinins and hydroxyl
radical
You Shuei Lin, Yu Ru Kou)
Institute of Physiology, School of Medicine and Life Science, National Yang-Ming UniÕersity, Shih-Pai, Taipei 11221, Taiwan
Received 18 October 1999; received in revised form 3 February 2000; accepted 8 February 2000
Abstract
We studied the mechanisms underlying the wood smoke-induced acute airway injury in 120 anaesthetized guinea pigs. Five minutes
after airway exposure, various doses of wood smoke produced a dose-dependent increase in Evans blue dye contents at all airway levels
measured. Additionally, inhaled wood smoke produced submucosal edema of the trachea and bronchus, and peribronchial edema. These
acute airway responses were nearly abolished by pretreatment with CP-96,345 alone wa tachykinin NK receptor antagonist; Ž2S,3S.-cis-
1
2
-Ždiphenylmethyl.-N-ŽŽ2-methoxyphenyl.-methyl.-1-azabicycloŽ2.2.2..-octan-3-aminex or with a combination of CP-96,345 and dimeth-
ylthiourea Ža hydroxyl radical scavenger., and were attenuated by pretreatment with dimethylthiourea alone, yet were not affected by
pretreatment with SR-48,968 wa tachykinin NK receptor antagonist; ŽS.-N-methyl-NŽ4-Ž4-acetylamino-4-phenylpiperidino.-2-Ž3,4-di-
2
chlorophenyl.-butyl.benzamidex, with a combination of CP-96,344 and SR-48,965 Žinactive enantiomers., with MK-886 wa leukotriene
biosynthesis inhibitor; L-663,536Ž3-Ž1-Ž4-chlorobenzyl.-3-t-butyl-thio-5-isopropylindol-2-yl.-2,2-dimethylpropanoic acidx, with indo-
G
methacin Ža cyclooxygenase inhibitor., or with N -nitro-L-arginine methyl ester Ža nitric oxide ŽNO. synthase inhibitor.. The activity of
airway neutral endopeptidase Žan enzyme for tachykinin degradation. was not influenced by wood smoke at 5-min post-exposure. We
conclude that both endogenous tachykinins and hydroxyl radical play an important role in producing smoke-induced acute airway plasma
exudation and airway edema in guinea pigs. The contribution of tachykinins to these neurogenic responses is mediated via the activation
of tachykinin NK1 receptors and partly via a hydroxyl radical mechanism, and is not associated with inactivation of neutral
endopeptidase. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Wood smoke; CP-96,345; Tachykinin NK1 receptor; Tachykinin NK2 receptor
1
. Introduction
an important component of airway inflammation ŽBarnes,
996a.. Tissue edema may possibly obstruct the airways
and contribute to ventilation–perfusion mismatching, res-
piratory insufficiency, and lung atelectasis observed in fire
victims ŽCrapo, 1981; Traber and Herndon, 1986.. While
these two smoke-induced airway responses are well recog-
nized, their pathophysiological mechanisms remain largely
unclear.
1
Smoke inhalation injury is one of the most severe
complications associated with fire-related disasters ŽCrapo,
981.. Inhalation of toxic smoke is known to initially
cause acute airway injury in humans ŽTraber and Herndon,
986. and laboratory animals ŽHales et al., 1989; Barrow
1
1
et al., 1990; Traber et al., 1992; LaLonde et al., 1994;
Sakurai et al., 1998.. Among several symptoms, airway
microvascular leakage and airway edema are two cardinal
signs of smoke-induced airway injury ŽTraber and Hern-
don, 1986; Traber et al., 1992; Barrow et al., 1990..
Tachykinins, such as substance P and neurokinin A, are
proinflammatory sensory neuropeptides released locally
from airway afferent C-fibre nerve endings in response to
various chemical irritants ŽSolway and Leff, 1991; Barnes,
Plasma exudation resulting from microvascular leakage is
1
996a.. Once released, tachykinins can cause several neu-
rogenic airway responses including plasma exudation and
tissue edema ŽSolway and Leff, 1991; Barnes, 1996a;
Advenier et al., 1997.. Their effects are mediated mainly
via activation of tachykinin NK and NK receptors ŽSol-
)
Corresponding author. Tel.: q886-2-2826-7086; fax: q886-2-2826-
4
0
049.
E-mail address: yrkou@ym.edu.tw ŽY.R. Kou..
1
2
014-2999r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S0014-2999Ž00.00126-6

1
40
Y.S. Lin, Y.R. KourEuropean Journal of Pharmacology 394 (2000) 139–148
way and Leff, 1991; Barnes, 1996a; Advenier et al., 1997..
tion of chloralose Ž100 mgrkg; Sigma, St. Louis, MO,
USA. and urethane Ž500 mgrkg; Sigma.. The carotid
artery and jugular vein were cannulated for recording
arterial blood pressure and for intravenous administration
of pharmacological agents, respectively. During the course
of the experiments, supplemental doses of chloralose Ž20
mgrkgrh. and urethane Ž100 mgrkgrh. were adminis-
The tachykinins released are rapidly degraded by neutral
endopeptidase, and inhibition of this enzyme may exagger-
ate the airway effects of tachykinins ŽSolway and Leff,
1
991; Barnes, 1996a.. We previously reported that inhaled
wood smoke stimulates lung vagal C-fibre afferent nerve
endings ŽLai and Kou, 1998. and inactivates airway neutral
endopeptidase ŽHsu et al., 1998a, 2000.. This raises the
possibility that tachykinin mechanisms may participate in
smoke-induced airway responses. Indeed, endogenous
tachykinins have been shown to be responsible for produc-
ing acute bronchoconstriction ŽHsu et al., 1998b. and
airway hyperreactivity ŽHsu et al., 1998a. following wood
smoke inhalation. However, the importance of tachykinins
and their degradation enzyme in the pathogenesis of
smoke-induced acute airway plasma exudation and airway
edema has not been elucidated.
In addition to tachykinins, inhalation of toxic smoke
may elevate the oxygen radical burden in the airways
ŽPryor, 1992; Youn et al., 1992., increase the release of
other chemical mediators such as lipoxygenase Ži.e. leuko-
trienes. and cyclooxygenase products Ži.e. prostaglandins
and thromboxane. ŽTraber and Herndon, 1986; Quinn et
al., 1990., and activate nitric oxide ŽNO. synthase to
generate NO ŽSoejima et al., 1999.. Among the major
types of oxygen radicals, the hydroxyl radical ŽPOH. is
tered to maintain the abolition of pain reflexes induced by
pinching the animal’s hindpaw. The animals were fixed in
a supine position and the trachea was cannulated below the
larynx with a short tracheal cannula via a tracheotomy,
through which the animals were ventilated with a rodent
respirator Žmodel 683, Harvard Apparatus, South Natick,
MA, USA. at a constant rate of 60 breathsrmin. Tidal
volume was adjusted according to the body weight of each
animal Ž10 mlrkg. and was kept constant in each experi-
ment. The animals were then paralyzed with an intra-
venous injection of pancuronium bromide Ž0.1 mgrkgrh;
Organon Teknika, Boxtel, Holland.. Throughout the exper-
iment, the body temperature of the animals was maintained
at ;368C by means of a servo heating blanket. All
protocols were in accordance with the guidelines for the
care and use of laboratory animals published by the Na-
tional Institutes of Health, Bethesda, MD, USA and were
approved by the Committee of the National Science Coun-
cil, Taipei, Taiwan.
extremely reactive and NO has many biological functions.
Lipoxygenase and cyclooxygenase products are arachi-
donate metabolites. These two categories of chemical me-
diators are known to possess potent effects on the produc-
tion of airway plasma exudation and airway edema ŽChung
2
.2. Generation and deliÕery of wood smoke
The electric furnace and the methods for generating
wood smoke were described in detail in our previous
report ŽKou and Lai, 1994.. In brief, 100 g of dry wood
dust Žlauan wood. was thermally decomposed in a furnace
Žmodel 101, Nan Jou, Taipei, Taiwan. at a core tempera-
ture maintained at 500"88C for 5 min and the effluent
smoke was collected in a 25-l plastic balloon attached to
the furnace outlet. Wood smoke generated by this method
contains approximately 1.5% O , 15% CO , 24% CO, and
et al., 1990; Barnes, 1996b; Barnes et al., 1998; Lei et al.,
1
996., and have been implicated in the pathogenesis of
inhalation injury ŽQuinn et al., 1990; Youn et al., 1992;
Loick et al., 1993; Soejima et al., 1999.. Nevertheless, the
role of POH, arachidonate metabolites, and NO in the
development of smoke-induced acute airway plasma exu-
dation and airway edema is not completely understood.
The present study with anaesthetized guinea pigs was
undertaken to determine Ž1. the dose–response relationship
and time course of acute airway plasma exudation pro-
duced by inhaled wood smoke, Ž2. the relative contribu-
tions of tachykinins, POH, arachidonate metabolites, and
NO to smoke-induced airway exudative and edematous
responses, and Ž3. whether these acute airway responses
2
2
2
5 mgrl particulates ŽKou et al., 1995.. Immediately after
smoke generation, the plastic balloon containing the fresh
smoke was attached to the inspiratory inlet of the respira-
tor via a 3-way stopcock. Wood smoke at a temperature of
;
258C was delivered into the lungs when the 3-way
stopcock was turned to connect the respirator with the
balloon. Before each smoke challenge, the lungs were
hyperinflated Ž4=tidal volume. to establish a constant
are associated with a smoke-induced reduction in airway
neutral endopeptidase activity.
volume history. To avoid contamination, the expired smoke
was drawn into a fume hood via a suction line.
2
. Materials and methods
2
.3. Measurements of plasma exudation
2
.1. Animal preparation
The magnitude of plasma exudation was quantified by
measuring the extravasation of Evans blue dye, using a
method described previously ŽEvans et al., 1988.. Evans
blue dye Ž25 mgrkg. was injected intravenously 1 min
Male Hartley guinea pigs Žbody weight, 307"4 g;
ns120. were anaesthetized with an intraperitoneal injec-

Y.S. Lin, Y.R. KourEuropean Journal of Pharmacology 394 (2000) 139–148
141
before the air or smoke challenge. Five, 60, or 120 min
after airway challenge, the thorax was quickly opened and
order to avoid possible artifacts dependent on differences
in airway size. For each histological section, structures at
three different areas were randomly selected and their
degrees of tissue edema were averaged.
the lungs were inflated, using a pressure of 5 cm H O.
2
Both the right and left atria were incised. A blunt, 12-gauge
needle was inserted into the pulmonary artery via a ven-
triculotomy. The heart was clamped and the pulmonary
circulation was perfused with 25 ml of saline at a pressure
of 30 mm Hg in order to remove the intravascular dye.
Subsequently, the left ventricle was opened and the sys-
temic circulation was perfused with 200 ml of saline at a
pressure of 120 mm Hg. Following vascular perfusion, the
left bronchus was ligated and the trachea was cut longitu-
dinally. The airways and lungs were then excised and
separated into left and right portions; the left portion was
immediately processed for histological preparations. The
connective tissues, vasculature, and parenchyma of the
right lung were gently scraped off with a blunt scalpel
until only the airway tissue was left. The right airway
tissues were then sectioned and subdivided into three
levels: the trachea, main bronchus, and intrapulmonary
airways. Both the airway and scraped parenchyma tissues
were blotted dry and weighed. Tissue Evans blue dye was
extracted by incubation in 2 ml of formamide ŽSigma. at
2
ity
.5. Determination of airway neutral endopeptidase actiÕ-
The activity of airway neutral endopeptidase was deter-
mined as described previously ŽHsu et al., 1998a.. Briefly,
frozen airway tissues were thawed and minced in tubes
containing 50 mM Tris, pH 7.4. The tissues were then
sonicated for 30 s at 48C and centrifuged at 17,500=g for
1
5 min. The supernatant was removed for analysis. In the
presence of substrate, the amount of 2-naphthylamine re-
leased by the tissues was determined using a spectro-
photometer ŽU-100, Hitachi. at 530 nm. The phosphorami-
don-inhibitable neutral endopeptidase specific activity was
expressed as nmolrmg of proteinrh.
2
.6. Pharmacological pretreatments
4
08C for 24 h and its concentration was measured by light
A tachykinin NK1 receptor antagonist wCP-96,345, 5
absorbance ŽMultiscan spectrophotometer, U-100, Hitachi,
Japan. at 620 nm. The tissue content of Evans blue dye
was calculated by interpolation on a standard curve of dye
concentrations in the range of 0.05–10 mgrml and was
expressed as ng dyermg of wet tissue.
mgrkg, 5 mgrml; Ž2S,3S.-cis-2-Ždiphenylmethyl.-N-ŽŽ2-
methoxyphenyl.-methyl.-1-azabicycloŽ2.2.2.. -octan-3-ami-
ne; Pfizer, Groton, CT, USAx, an inactive enantiomer of
CP-96,345 wCP-96,344, 5 mgrkg, 5 mgrml; Ž2 R,3R.-cis-
2-Ždiphenylmethyl.-N-ŽŽ2-methoxyphenyl.-methyl.-1-aza-
bicycloŽ2.2.2..-octan-3-amine; Pfizerx, a tachykinin NK
2
2
.4. Histological preparations and examinations
receptor antagonist wSR-48,968, 2 mgrkg, 1 mgrml; ŽS.-
N- methyl-NŽ4-Ž4-acetylamino-4-phenylpiperidino. -2- Ž3,4-
dichlorophenyl.-butyl.benzamide; Sanofi Research, Mont-
pellier, Francex, and an enantiomer of SR-48,968 wSR-
48,965, 2 mgrkg, 1 mgrml; ŽR.-N-methyl-NŽ4-Ž4-acety-
lamino-4-phenylpiperidino.-2-Ž3,4-dichlorophenyl.-
butyl.benzamide; Sanofi Researchx were each dissolved in
isotonic saline. A NO synthase inhibitor ŽL-NAME, 10
Immediately after their excision, the left portions of the
airway and lung tissues were fixed by immersion in a
buffered neutral formalin solution for 48 h. Tissue speci-
mens were embedded in paraffin and were cut transversely
into 5 mm thick sections which were subsequently stained
with hematoxylin and eosin. For each animal, one histolog-
ical section was obtained from the trachea and also from
the main bronchus, while two sections were obtained from
the lung tissues. These sections were examined and photo-
graphed with a light microscope ŽAxioskop MC 100, Zeiss,
Germany. at a magnification of 200= or 400=. The
degree of submucosal edema in the trachea and bronchus
was determined from the ratio of the submucosal layer
thickness to the epithelial thickness Žsubmucosal edema
index.. The degree of peribronchial edema was estimated
by a method modified from that reported previously ŽGarcia
et al., 1994.. In brief, photographs of histological sections
G
mgrkg; N -nitro-L-arginine methyl ester; Sigma. was also
dissolved in isotonic saline to a concentration of 10 mgrml.
A leukotriene biosynthesis inhibitor wMK-886, 10 mgrkg;
L-663,536Ž3-Ž1-Ž4-chlorobenzyl.-3-t-butyl-thio-5-isopro-
pylindol-2-yl.-2,2-dimethylpropanoic acid.; Calbiochem,
CA, USAx was first dissolved in 200 ml dimethyl sulfoxide
ŽSigma. and then diluted with saline to a final concen-
tration of 10 mgrml. A cyclooxygenase inhibitor Žin-
domethacin, 10 mgrkg; Sigma. was first dissolved in
polyethylene glycol and then diluted in a 1:1 ratio with
saline to a final concentration of 10 mgrml. All the
above-mentioned drugs were injected as boluses into the
vein. A POH scavenger Ždimethylthiourea, 4.5 grkg;
Sigma. was dissolved in isotonic saline to a concentration
of 0.75 grml and was slowly infused into the vein for a
period of 20 min with an infusion pump ŽSage 367,
Cambridge, MA, USA.. We used two criteria to judge
were superimposed with a transparency comprising 3620
2
points in 605 cm . Points falling on the peribronchial cuff
of the connective tissue were counted and were considered
as an index of the area occupied by interstitial edema. The
number of points falling on the peribronchial cuff was
divided by the perimeter of the corresponding airway to
give an index of tissue edema. This procedure was done in
whether the doses of compounds used were sufficient

1
42
Y.S. Lin, Y.R. KourEuropean Journal of Pharmacology 394 (2000) 139–148
enough. First, we looked for the doses of these drugs that
are equal to or higher than those previously used by other
investigators ŽEvans et al., 1988; Guhlmann et al., 1989;
smoke were investigated at 5-min post-smoke exposure in
eight groups of animals pretreated with either CP-96,345
alone, SR-48,968 alone, dimethylthiourea alone, CP-96,345
and dimethylthiourea in combination, CP-96,344 and SR-
48,965 in combination, MK-886, indomethacin, or L-
NAME 10–15 min before smoke challenge. In Study 3,
one group of animals received a sham air challenge, while
the other group received 40 breaths of wood smoke. Five
minutes after the air or smoke exposure, the animals were
killed by an overdose of anaesthetics. Then the airway
tissues including the tracheal segment below the tip of the
tracheal cannula and the main stem bronchus were quickly
removed, washed with isotonic saline, and stored at y708C
for analysis of neutral endopeptidase activity.
Sakamoto et al., 1994; Lei et al., 1996; Kageyama et al.,
1
997. in studies of lung pathophysiology induced by other
experimental conditions in guinea pigs. Second, in the
preliminary study, we doubled or tripled the dose if any
compound failed to influence the smoke-induced airway
responses.
2
.7. Experimental procedures
A total of 120 animals were randomly and equally
divided into 20 groups. In Study 1, airway exudative
responses to air and to 10, 20, or 40 breaths of wood
smoke were investigated at 5-min post-smoke exposure in
four groups of animals. Additionally, airway exudative
responses to air and to 40 breaths of wood smoke were
investigated at 60- or 120-min post-smoke exposure in
another four groups of animals. In Study 2, airway exuda-
tive and edematous responses to air or 40 breaths of wood
smoke were investigated at 5-min post-smoke exposure in
two groups of animals to establish the airrvehicle and
smokervehicle controls, respectively. Additionally, airway
exudative and edematous responses to 40 breaths of wood
2
.8. Statistical analysis
Results of Evans blue dye extravasation, submucosal
edema index, and peribronchial edema index were ana-
lyzed by a one-factor analysis of variance followed by
Fisher’s least significant difference procedure when appro-
priate. Data for airway neutral endopeptidase activity were
analyzed with Student’s t-test. P-0.05 was considered
significant. All data are presented as means "S.E.
Fig. 1. Evans blue ŽEB. dye extravasation of trachea, bronchus, intrapulmonary airways, and lung parenchyma induced by air or various doses of wood
smoke measured at different times Ž5, 60, and 120 min. post-smoke exposure. SM10, SM20, and SM40 represent 10, 20, and 40 breaths of smoke,
a
b
U
respectively. significantly different from response to air at 5 min; significantly different from response to SM10 at 5 min; significantly different from
a
@
the corresponding response to air at 60 or 120 min; significantly different from the corresponding response to air or SM40 at 5 min; significantly
different from the corresponding response to air or SM40 at 60 min. Data in each group are means"S.E. for six animals.

Y.S. Lin, Y.R. KourEuropean Journal of Pharmacology 394 (2000) 139–148
143
3
. Results
of wood smoke at all airway levels and in lung parenchyma
still persisted and were greater than those measured at
5-min post-airway exposure ŽFig. 1.. The responses to 40
3
.1. Dose–response relationship and time course of airway
exudatiÕe response
breaths of wood smoke with vascular perfusion 5-min
post-airway exposure were then taken as the standard
responses for subsequent studies in animals pretreated with
various pharmacological agents or a saline vehicle.
Five-minute post-airway exposure and then after vascu-
lar perfusion, wood smoke acutely and markedly produced
airway plasma exudation at all airway levels measured, as
evidenced by an increase in Evans blue dye content in
these tissues ŽFig. 1.. When various tidal breaths of wood
smoke were delivered, the airway exudative responses
were dose-dependent. Statistical analysis revealed that de-
livery of 20 or 40 breaths of wood smoke significantly
increased dye contents at all airway levels, and that deliv-
ery of 10 breaths of wood smoke significantly increased
dye contents in the trachea and bronchus ŽFig. 1.. Further-
more, there was no significant difference between the
airway exudative responses induced by 20 and by 40
breaths of wood smoke, but both were significantly greater
than the response induced by 10 breaths of wood smoke
ŽFig. 1.. In contrast, only delivery of up to 40 breaths of
3
.2. Effects of pharmacological pretreatments on airway
exudatiÕe and edematous responses
In response to delivery of 40 breaths of wood smoke,
the airways of animals pretreated with saline vehicle fully
displayed Evans blue dye extravasation ŽFig. 2.. Addition-
ally, histological examinations revealed substantial submu-
cosal edema in the trachea ŽFig. 3B1. and bronchus ŽFig.
3B2., and apparent peribronchial edema ŽFig. 3B3.. How-
ever, neither major morphological changes in the alveolar
region nor alveolar flooding were noted. In animals pre-
treated with CP-96,345 alone, dimethylthiourea alone, or
CP-96,345 and dimethylthiourea in combination, both the
airway exudative ŽFig. 2. and edematous responses ŽFig. 4.
wood smoke induced a small but significant increase in
dye content in lung parenchyma ŽFig. 1.. When the time of
vascular perfusion was delayed to either 60- or 120-min
to inhaled wood smoke were significantly attenuated. In
post-smoke exposure, the exudative responses to 40 breaths
sharp contrast, in animals pretreated with SR-48,968 alone,
Fig. 2. Evans blue ŽEB. dye extravasation of trachea, bronchus, intrapulmonary airways, and lung parenchyma induced by air or 40 breaths of wood smoke
measured at 5 min post-smoke exposure in animals pretreated with saline vehicle, CP-96,345 alone ŽCP; 5 mgrkg., SR-48,968 alone ŽSR; 2 mgrkg.,
dimethylthiourea alone ŽDMTU; 4.5 grkg., CP-96,345 Ž5 mgrkg. and DMTU Ž4.5 grkg. in combination ŽCPqDMTU., CP-96,344 Ž5 mgrkg. and
a
SR-48,965 Ž2 mgrkg. in combination ŽCPIqSRI., MK-886 Ž10 mgrkg., indomethacin ŽIndo.; 10 mgrkg., or
L
-NAME Ž10 mgrkg.. significantly
b
different from response to air in animals pretreated with saline vehicle; significantly different from response to smoke in animals pretreated with saline
vehicle; significantly different from response to smoke in animals pretreated with CP. Data in each group are means"S.E. for six animals.
c

1
44
Y.S. Lin, Y.R. KourEuropean Journal of Pharmacology 394 (2000) 139–148
Fig. 3. Light micrographs of epithelial and submucosal space of trachea ŽA1 and B1. and bronchus ŽA2 and B2., and peribronchial cuff ŽA3 and B3. in two
guinea pigs. Tissues were excised 5 min post-airway exposure to air ŽA1, A2, and A3. or 40 breaths of wood smoke ŽB1, B2, and B3.. E, epithelium; S,
submucosal space; PB, peribronchial cuff. Magnification: 400= in A1, A2, B2, and B2; 200= in A3 and B3. Note that both submucosal space and
peribronchial cuff were greatly enlarged in tissues exposed to smoke.
CP-96,344 and SR-48,965 in combination, MK-886, indo-
methacin, or L-NAME, these smoke-induced airway re-
sponses were not significantly affected ŽFigs. 2 and 4..
Furthermore, smoke-induced exudative responses at all
airway levels ŽFig. 2. and submucosal edema of the tra-
chea and bronchus ŽFig. 4. were moderately reduced by
dimethylthiourea to magnitudes that were still significantly
different from the air controls, and were greatly reduced by
CP-96,345 to magnitudes that were not significantly differ-
ent from the air controls ŽFig. 2.. Pretreatment with CP-
96,345 and dimethylthiourea in combination did not fur-
ther attenuate either of the smoke-induced airway re-
sponses, as compared to pretreatment with CP-96,345 alone
ŽFigs. 2 and 4.. Finally, the small but significant increase
in Evans blue dye content in lung parenchyma was also
abolished by pretreatment with CP-96,345 alone or in
combination with dimethylthiourea, but was not affected
by other pharmacological interventions ŽFig. 2..

Y.S. Lin, Y.R. KourEuropean Journal of Pharmacology 394 (2000) 139–148
145
Fig. 4. Increases in ratio of submucosal thickness to epithelial thickness, and peribronchial edema index induced by air or 40 breaths of wood smoke in
animals pretreated with saline vehicle, CP-96,345 alone ŽCP; 5 mgrkg., SR-48,968 alone ŽSR; 2 mgrkg., dimethylthiourea alone ŽDMTU; 4.5 grkg.,
CP-96,345 Ž5 mgrkg. and DMTU Ž4.5 grkg. in combination ŽCPqDMTU., CP-96,344 Ž5 mgrkg. and SR-48,965 Ž2 mgrkg. in combination
a
ŽCPIqSRI., MK-886 Ž10 mgrkg. , indomethacin ŽIndo.; 10 mgrkg., or
L
-NAME Ž10 mgrkg.. significantly different from response to air in animal
b
c
pretreated with saline vehicle; significantly different from response to smoke in animals pretreated with saline vehicle; significantly different from
response to smoke in animals pretreated with CP. Data in each group are means"S.E. for six animals. See text for details of measurement of peribronchial
edema index.
3
.3. Effects of wood smoke on airway neutral endopepti-
parenchyma and caused no obvious histological changes in
the alveolar region or alveolar flooding. These effects are
similar to those reported from clinical and animal studies
ŽMoylan et al., 1972; Traber and Herndon, 1986; Barrow
et al., 1990; Sakurai et al., 1998., in which airway injury
dase actiÕity
The activity of airway neutral endopeptidase measured
in tissues excised 5 min after delivery of 40 breaths of
wood smoke Ž47.2"8.4 nmolrmg of proteinrh. was not
significantly different from that in tissues excised at the
same time after sham air challenge Ž45.2"4.2 nmolrmg
of proteinrh; P)0.05, ns6..
was prominent within the first few hours after toxic smoke
exposure, while lung parenchyma edema occurred with a
much longer delay from several hours to days after inhala-
tion injury. Furthermore, the present study showed that
dramatic pathophysiological changes in the airways occur
as rapidly as a few minutes after smoke inhalation. In
addition to the dose-dependent relationship of the smoke-
induced increase in dye content in tissues, delivery of 10
breaths of wood smoke significantly increased dye con-
tents only in the trachea and bronchus, whereas delivery of
20 or 40 breaths of wood smoke significantly increased
dye contents at all airway levels. Furthermore, only deliv-
ery of up to 40 breaths of wood smoke induced a signifi-
cant increase in dye content in lung parenchyma. There-
fore, it appears that a greater amount of wood smoke may
damage a greater area of airway tissues, from the large
airways to the lung parenchyma.
4
. Discussion
We demonstrated that, in anaesthetized guinea pigs,
delivery of wood smoke acutely induced airway plasma
exudation and airway edema. The former response, lasting
for )2 h, was evidenced by a large dose-dependent
increase in Evans blue dye content in tissues and may have
resulted from an increase in airway microvascular perme-
ability. The latter response was revealed histologically by
an enlargement of the submucosal space and peribronchial
cuff and was probably due to the accumulation of exuda-
tive fluid and macromolecules within airway tissues. In
contrast, only delivery of the largest dose of wood smoke
produced a slight increase in dye content in lung
In this study, pretreatment with CP-96,345 Ža tachykinin
NK receptor antagonist. nearly abolished both the airway
1
exudative and edematous responses to inhaled wood smoke,

1
46
Y.S. Lin, Y.R. KourEuropean Journal of Pharmacology 394 (2000) 139–148
whereas pretreatment with either SR-48,968 Ža tachykinin
NK2 receptor antagonist. or a combination of CP-96,344
and SR-48,965 Žinactive enantiomers. failed to do so.
administration of a scavenger for oxygen radicals ŽKimura
et al., 1988., an enzyme that metabolizes oxygen radicals
ŽNguyen et al., 1995., or an iron chelator that suppresses
the production of oxygen radicals ŽLaLonde et al., 1994..
The present findings indicate that oxygen radicals actually
participate in the very early process of inhalation injury.
Regardless of the time course of their participation, there is
little doubt that oxygen radicals play a contributory role in
the pathogenesis of smoke inhalation injury ŽPryor, 1992..
Likewise, oxygen radicals have also been largely impli-
cated in airway andror lung injury induced by various
oxidant irritants such as cigarette smoke and ozone ŽPryor,
1992; Youn et al., 1992; Lei et al., 1996.. The source of
the POH that produces the acute airway responses ob-
served in this study remains unclear, but wood smoke may
be one possible origin. The gas phase of wood smoke is
known to contain high concentrations of free radicals and
radical precursors which are formed during combustion
ŽPryor, 1992.. These free radicals and their precursors may
continuously generate oxygen radicals either in the smoke
or upon reaching the airways ŽTraber and Herndon, 1986;
Pryor, 1992; LaLonde et al., 1994.. The other possible
origin is that POH may be formed and released endoge-
nously by certain inflammatory cells when they are acti-
vated by smoke inhalation ŽTraber and Herndon, 1986..
Indeed, there is substantial evidence that oxygen radical
activity is increased in airways and lungs exposed to toxic
smoke ŽDemling et al., 1994, 1995..
Despite the fact that their contributions are clear, the
exact mechanisms by which tachykinins and POH partici-
pate in the development of smoke-induced acute airway
plasma exudation and airway edema remain unclear. Both
tachykinins and POH are known to have direct injurious
effects on the microvasculature ŽRubanyi, 1988; Chung et
al., 1990; Barnes, 1996a.. Furthermore, tachykinins, acting
as vasodilators, may also increase bronchial blood flow
ŽSolway and Leff, 1991; Barnes, 1996a., which may facili-
tate airway plasma exudation ŽChung et al., 1990.. In fact,
smoke-induced acute airway injury is usually accompanied
by a large increase in bronchial blood flow ŽKramer et al.,
1989; Traber et al., 1992.. Therefore, tachykinins and POH
might act independently in different airway tissues to
produce these two airway responses. However, in the
present study, pretreatment with CP-96,345 and dimeth-
ylthiourea in combination did not seem to further attenuate
these two airway responses, as compared to pretreatment
with CP-96,345 alone. Accordingly, it appears that there is
an overlap of functional contributions of tachykinins and
POH to smoke-induced airway exudative and edematous
responses. POH may inhibit airway neutral endopeptidase
and exaggerate the airway effects of tachykinins ŽSolway
and Leff, 1991; Barnes, 1996a.. This is unlikely, however,
These results suggest that endogenous tachykinins play an
essential role in the production of smoke-induced acute
airway plasma exudation and airway edema, and that the
contribution of tachykinins is primarily mediated through
the activation of tachykinin NK1 receptors. Neurogenic
airway responses, involving C-fibre afferents and their
containing tachykinins, have been well documented as
major consequences of airway assault by various inhaled
irritants such as cigarette smoke or ozone ŽSolway and
Leff, 1991; Barnes, 1996a; Lei et al., 1996.. In a recent
study ŽHsu et al., 1998b., we found that the activation of
tachykinin NK2 receptors by endogenous tachykinins is
responsible for producing the bronchoconstriction induced
by inhaled wood smoke. Thus, our observations are in
good agreement with the current viewpoint regarding neu-
rogenic airway responses, that plasma extravasation and
airway smooth muscle contraction are predominantly me-
diated through the activation of tachykinin NK and NK2
1
receptors, respectively ŽSolway and Leff, 1991; Barnes,
1
996a; Advenier et al., 1997.. The contribution of
tachykinins to the observed airway exudative and edema-
tous responses may originate from an increase in their
release from airway afferent C-fibre nerve endings when
wood smoke stimulates these pulmonary receptors ŽKou et
al., 1995; Lai and Kou, 1998. andror from smoke-induced
inactivation of airway neutral endopeptidase, the major
enzyme for tachykinin degradation ŽSolway and Leff, 1991;
Barnes, 1996a.. We previously reported that the activity of
airway neutral endopeptidase in guinea pigs is reduced 15
min after inhalation of 15 breaths of wood smoke ŽHsu et
al., 1998a, 2000.. However, we found no evidence of the
involvement of neutral endopeptidase inactivation in the
acute airway exudative and edematous responses observed
in the present study, because neutral endopeptidase activity
was not significantly affected in airway tissues excised 5
min after delivery of 40 breaths of wood smoke, a time
when these airway responses occurred. In a follow-up
study, we found that the activity of airway neutral en-
dopeptidase in guinea pigs was reduced 120 min after
inhalation of 40 breaths of wood smoke ŽY.S. Lin and
Y.R. Kou, unpublished data.. Therefore, it would be as-
sumed that the time after smoke insult, but not the amount
of wood smoke, could be a more important factor for the
inactivation of airway neutral endopeptidase by inhaled
wood smoke.
We further demonstrated that pretreatment with dimeth-
ylthiourea, an effective POH scavenger, moderately but
significantly attenuated both the airway exudative and
edematous responses to inhaled wood smoke, suggesting
that POH is actively involved in producing these acute
airway responses. Several investigators have shown that, in
a sheep model, airway andror lung injury assessed several
hours after toxic smoke inhalation can be ameliorated by
because airway neutral endopeptidase was not inhibited in
this study. A plausible explanation for the overlap of
contributions is that tachykinins and POH are interrelated
in their release. For example, it has been shown that POH

Y.S. Lin, Y.R. KourEuropean Journal of Pharmacology 394 (2000) 139–148
147
is the major chemical factor responsible for the stimulation
of bronchopulmonary C-fibre afferents by inhaled wood
smoke ŽKou et al., 1997; Lai and Kou, 1998.. Thus,
ling through the bronchial circulation ŽHales et al., 1989;
Traber et al., 1992; Sakurai et al., 1998.. The proinflam-
matory role of tachykinins in the pathogenesis of delayed
smoke-induced lung edema is largely unknown and thus
requires further investigation.
In conclusion, these results showed that both endoge-
nous tachykinins and POH, but not arachidonate metabo-
lites or NO, play an important role in producing smoke-in-
duced acute airway plasma exudation and airway edema in
guinea pigs. Additionally, the contribution of tachykinins
to these two acute neurogenic airway responses is primar-
scavenging POH by dimethylthiourea would reduce both
the magnitude of the stimulation of C-fibre nerve endings
by wood smoke and the amount of the subsequent release
of tachykinins available for producing these two airway
responses. However, dimethylthiourea presumably could
not interfere with the part of the responses caused by the
release of tachykinins due to smoke-related stimuli other
than POH. Additionally, it has been demonstrated that
tachykinins have the ability to increase the production of
oxygen radicals from certain lung cells ŽBrunelleschi et al.,
ily mediated via the activation of tachykinin NK recep-
1
tors, is mediated in part by a POH mechanism, and is not
associated with inactivation of airway neutral endopepti-
dase.
1
990; Murris-Espin et al., 1995.. In this case, blocking
tachykinin receptors by their antagonist may possibly elim-
inate the contributions from both tachykinins and POH.
Inhaled toxic smoke can increase the release of cyclo-
oxygenase and lipoxygenase products ŽTraber and Hern-
don, 1986; Quinn et al., 1990., and activate NO synthase
to generate NO ŽSoejima et al., 1999.. It is known that
these arachidonate metabolites and NO can increase air-
way microvascular permeability ŽChung et al., 1990;
Barnes, 1996b; Barnes et al., 1998.. However, pretreat-
ment with either indomethacin Ža cyclooxygenase in-
hibitor., MK-886 Ža leukotriene biosynthesis inhibitor., or
L-NAME Ža NO synthase inhibitor. failed to modify the
Acknowledgements
We acknowledge Drs. Tien Huan Hsu National Chung
Ž
Hsing University, Taiwan and Hung-Tu Huang National
Sun Yat-Sen University, Taiwan for their help with histo-
logical preparations and examinations. We thank Mr. D. P.
Chamberlin for editorial assistance. We are also grateful to
.
Ž
.
Dr. J. A. Lowe, III Pfizer, Groton, CT, USA for provid-
ing CP-96,345 and CP-96,344 and to Dr. X. Emonds-Alt
Ž
.
acute airway exudative and edematous responses to wood
smoke, suggesting that these arachidonate products and
NO are unlikely to play a vital role. It has been shown that
pulmonary edema observed 6 h after smoke inhalation in
rats ŽShinozawa et al., 1986. and 3 h after smoke inhala-
tion in sheep ŽQuinn et al., 1990. can be attenuated by
Ž
.
Sanofi, Montpellier, France for providing SR-48,968 and
SR-48,965. This study was supported by the National
Science Council of the Republic of China Grants 89-2320-
B010-061-M41 and 89-2320-B010-043.
pretreatment with a cyclooxygenase inhibitor and a lipoxy-
genase inhibitor, respectively. Additionally, it has been
reported that the increase in airway blood flow and the
deterioration of arterial blood oxygenation observed 48 h
after smoke inhalation in sheep can be attenuated by a NO
synthase inhibitor ŽSoejima et al., 1999.. Thus, different
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