Vol. 91, Issue 6, 2595-2601, December 2001
Differences between inhaled and intravenous bronchial
challenge to detect O3-induced hyperresponsiveness
Bettina
Sommer,
Mario H.
Vargas*,
Jaime
Chavez,
Veronica
Carbajal,
Patricia
Segura, and
Luis M.
Montaño*
Departamento de Investigación en Asma, Instituto Nacional de
Enfermedades Respiratorias, and Departamento de
Farmacología, Facultad de Medicina, Universidad Nacional
Autónoma de México, CP 14080, México DF,
México
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ABSTRACT |
Ozone
(O3)-induced airway hyperresponsiveness in laboratory
animals is usually demonstrated through dose-response curves with inhaled or intravenous bronchoconstrictor agonists. However,
comparability of these two routes has not been well documented. Thus
guinea pig airway responsiveness to ACh and histamine was evaluated
16-18 h after O3 (3 parts/million, 1 h) or air
exposure by two plethysmographic methods (spontaneously breathing and
mechanically ventilated) and by two administration routes (inhalatory
or intravenous). We found that O3 caused airway
hyperresponsiveness to intravenous, but not to inhaled, agonists,
independent of the plethysmographic method used. Suitability of the
inhalatory route to detect airway hyperresponsiveness was corroborated
with inhaled ACh after an antigen challenge or extending O3
exposure to 3 h. Acetylcholinesterase activity was not modified
after O3 exposure in lung homogenates and blood samples.
Thus inhaled agonists were less effective to reveal the airway
hyperresponsiveness after an acute O3 exposure than
intravenous ones, at least for the 1-h exposure to 3 parts/million, and
this difference seems not to be related to an O3-induced
inhibition of the acetylcholinesterase activity.
ozone; airway hyperresponsiveness; dose-response curve; guinea pig
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INTRODUCTION |
EXPOSURE
TO OZONE (O3) is a widely used method to induce
airway hyperresponsiveness in different species, including guinea pigs
and humans (3, 15, 20). The increased
responsiveness is usually demonstrated through pharmacological methods,
i.e., the elaboration of dose-response curves with
bronchoconstrictor agents. In laboratory animals, the most common
ways to deliver these agents to the airways are the intravenous
(22, 23) and the inhalatory routes (14,
17). Although extensively used to demonstrate this
phenomenon, differences between inhaled and intravenously
administered agents have been very scantily investigated (18). This is an important issue because conclusions
derived from experiments using one route might not be comparable with those using the other one, unless equivalence of both routes was assessed. In this sense, Corddry et al. (8) have
demonstrated in dogs that whole lung resistance (RL) and
peripheral airway resistance (Raw) measurements detected the same
pattern of responsiveness to intravenous histamine (His), whereas they
yielded different results with nebulized His. In the present work, we
found that, at a certain O3 exposure magnitude, inhaled
agonists did not detect airway hyperresponsiveness as the intravenous
ones did, and some possible mechanisms for this difference are discussed.
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MATERIALS AND METHODS |
Animals and experimental design.
Male Hartley guinea pigs (450-500 g body wt), bred in conventional
conditions (filtered conditioned air, 21 ± 1°C, 50-70% humidity, sterilized bed) and fed with Purina pellets supplemented with
disinfected fresh alfalfa and sterilized water, were used. Airway
hyperresponsiveness to ACh and His was assessed by constructing dose-response curves. Two methods were used to measure the responses: plethysmography in spontaneously breathing (SB) animals and
plethysmography in mechanically ventilated (MV) animals. In either
method, bronchial challenges were accomplished by delivering the drugs
by nebulized (NEB) or intravenous (IV) route. Therefore, four main
groups of animals were integrated: SB/NEB challenge, SB/IV challenge,
MV/NEB challenge, and MV/IV challenge.
With regard to the SB/NEB group, the noninvasive nature of the
technique allowed us to perform two doses-response curves in the same
guinea pigs. In these animals, the first curve was done ~24 h before
the O3 or air (control group) exposure, and the second was
done 16-18 h after such exposure. In the remaining groups, only
one dose-response curve after air or O3 exposure was
performed in each animal. In addition, a group of sensitized guinea
pigs was submitted to a dose-response curve to inhaled ACh 24 h
before and 3 h after an antigenic challenge. Finally, separate
groups of guinea pigs with or without O3 exposure were used
to evaluate the acetylcholinesterase (AChE) activity in lung
homogenates and blood.
Plethysmography in spontaneously breathing animals.
Each guinea pig was introduced into a plethysmographic chamber designed
for unrestrained, freely moving animals (Buxco Electronics, Troy, NY).
This chamber is supplied with a constant air flow (10 ml/s) that does
not alter the respiratory signals. This methodology has been
comprehensively described elsewhere (5, 6, 12). Briefly,
the pressure inside the chamber was measured by a differential pressure
transducer (SCXL004DN, Sen Sym, Milpitas, CA) connected to a
preamplifier and continuously monitored through software (Buxco
Biosystem XA, version 1.1). Changes in box pressure represent the
difference between the thoracic expansion or contraction and the tidal
volume (air removed from or added to the chamber during inspiration or
expiration). The box pressure is differentiated to give a pseudoflow
signal, which is then analyzed by the software to give an index named
"enhanced pause" (Penh). The Penh value is obtained for each
respiration by the following formula
](/content/vol91/issue6/fulltext/2595/img001.gif)
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where TE is expiratory time, RT is relaxation time,
PEpeak is peak expiratory pressure, and
PIpeak is peak inspiratory pressure. Penh
values used in our study were obtained from averaging logged values
every 15 s. The software was adjusted to only include breaths with
a tidal value of 
1 ml, with a minimal inspiratory time of 0.15 s, a maximal inspiratory time of 3 s, and a maximal difference between inspiratory and expiratory volumes of 10%. A recent study in
mice (12) demonstrated that Penh had a close, positive
correlation with the measurement of total RL, which in turn
can reflect the Raw and/or the lung parenchymal tissue resistance.
Therefore, in this study, the Penh is considered as a total lung
resistance index (iRL), as has been previously proposed by
others and us (1, 6, 12, 21). Moreover, in the present
study, we used this plethysmographic technique to measure acute
responses to increasing concentrations of ACh or His. These responses
were characterized by a transient increase in iRL values,
spontaneously returning to baseline levels after ~10 min, implying
the reversibility of the RL increment, and thus pointing
out that airway obstruction was the main component of such increment.
This consideration is in agreement with the recent finding that airway
sensitivity to His observed in guinea pigs through Penh measurement was
very similar to the one obtained by specific Raw measurement
(7). Therefore, it is highly feasible that, in our
study, iRL mainly reflects Raw.
In this plethysmographic chamber, two routes of drug administration
were used: inhalatory (SB/NEB group) and intravenous (SB/IV group). In
the latter case, ACh or His was administered through a catheter placed
in the left jugular vein, and every guinea pig was put into a metallic
mesh that restricted its wide movements, limiting in this way the
possibility that the catheter could be pulled out by the animal.
Because of these last maneuvers (restriction of movements and invasive
procedures), comparison with freely moving animals might not be
appropriate. Therefore, an additional SB/NEB group was submitted to the
same maneuvers as for SB/IV.
Plethysmography for mechanically ventilated animals.
RL was measured through the isovolumetric method in a closed-chamber
plethysmograph (Buxco Electronics). Guinea pigs were anesthetized with
pentobarbital sodium (35 mg/kg ip), and the depth of anesthesia was
kept with hourly administration of additional doses of pentobarbital
(~9 mg/kg iv). Each animal received pancuronium bromide (0.06 mg/kg
iv) to avoid spontaneous respiratory movements. After the trachea was
cannulated, each animal was mechanically ventilated (model
50-1700, Harvard Apparatus) with a tidal volume of 10 ml/kg and 48 breaths/min. Right jugular vein and left carotid artery were cannulated
for drug administration and for arterial pressure recording through a
Beckman R-612 dynograph, respectively. A water-filled cannula was
positioned into the middle one-third of the esophagus to measure
intraesophageal pressure as a surrogate of intrapleural pressure.
Pressures obtained from the esophageal and tracheal cannulas were
recorded through a differential pressure transducer (SCXL004DN, Sen
Sym). Pressure inside the plethysmograph chamber was also
recorded through a differential pressure transducer. This last signal
was converted to a pseudoflow signal through software (Buxco Biosystem
XA, version 1.0). Finally, this software also calculated the
relationship between both parameters to obtain RL through
the formula RL = 
P/
, where P is pressure
change and is flow change.
Bronchial challenge.
Airway responsiveness was assessed through bronchial challenges using
inhaled or intravenous agonists in both spontaneously breathing and
mechanically ventilated guinea pigs.
The nebulized bronchial challenge in spontaneously breathing animals
was done in the plethysmographic chamber. Thus, after a baseline saline
nebulization (0.9% NaCl, 2 min), noncumulative increasing doses of ACh
(0.056-3.2 mg/ml) or His (0.018-3.2 mg/ml) were nebulized
during 2 min each. The interval between doses was ~5-12 min,
enough time to recover the basal iRL value. After each dose, the iRL was registered during 5 min, and the average
value was calculated. The procedure ended when the iRL
value after a certain dose was threefold or more of the basal
iRL value (obtained after saline). Nebulizations were done
by using a US-1 Bennett nebulizer (flow: 2 ml/min), with a mixed
particle size of 44% < 4 µm, 38% = 4-10 µm, and 18% > 10 µm (multistage liquid impinger, 20 l/min, Bukard Manufacturing). In
the case of mechanically ventilated guinea pigs, the nebulized agonists
were delivered to the inlet port of the ventilator via a plastic
reservoir, and in these last animals the exposure to nebulized agonist
lasted only 1 min.
With regard to the intravenous bronchial challenge, after a baseline
response to saline (0.1 ml iv of 0.9% NaCl) a dose-response curve for
ACh (0.18-56 µg/kg iv) or His (0.18-56 µg/kg iv) was constructed. After each dose, the maximum response (iRL in
spontaneously breathing guinea pigs, and RL in mechanically
ventilated) obtained at any averaged 10-breath period during the
following 3 min was registered. As described in the previous paragraph,
the dose-response curve was finished when a response was threefold the
baseline iRL or RL value.
O3 exposure.
Animals were placed in a Plexiglas exposure chamber (48 × 73 × 32 cm) where they were continuously exposed to 3 parts/million (ppm)
O3 during 1 h. O3 was produced by passing
a constant air flow (3 l/min) through an ozonizer (Puraqua-V,
Purificadores Eléctricos por Ozono, Mexico DF, Mexico), in which
an electric arc decomposes air into O3. The O3
concentration inside the exposure chamber was regulated by modifying
the voltage delivered to the ozonizer and monitored by an ultraviolet
O3 analyzer (model 1008 PC, Dasibi Environmental).
Sensitization procedure and antigenic challenge.
Guinea pigs were sensitized at day 0 by intraperitoneal
administration of 40 µg ovalbumin and 1 mg Al(OH)3 in 0.5 ml of saline solution. On day 8, the animals were nebulized
with 3 mg/ml ovalbumin in saline solution for 2 min, delivered by a
US-Bennett nebulizer. On day 15, guinea pigs were nebulized
again with 0.5 mg/ml ovalbumin in saline solution for 1 min. Animals
were studied on days 21-25. Antigenic challenge was
accomplished by delivering 0.5 mg/ml ovalbumin during 30 s by the
inhalatory route.
AChE activity.
The AChE activity was determined in guinea pig total lungs and blood
samples by using a colorimetric method based on the Ellman reaction
(9). Under deep anesthesia with an overdose of
pentobarbital, the guinea pig's chest was opened, and a heparinized
blood sample was obtained from cardiac puncture and refrigerated.
Afterwards, lungs were washed by injecting phosphate buffer through the
right ventricle, and all lung lobes were removed and stored at
20°C. On the next day, the lung tissue was homogenized in phosphate buffer (100 mg tissue/ml phosphate buffer) with a homogenizer (Polytron, Brinkmann Instruments, Westbury, NY). The homogenate was
centrifuged for 15 min at 3,000 g, and the supernatant was filtered (22 µm polytetrafluoroethylene filter). Three hundred microliters of supernatant were added to a cuvette containing 2.5 ml of
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; 0.32 mM) and 300 µl
phosphate buffer (64 mM). The background absorbance per minute of each
sample was measured at 405 nm at 25°C with a spectrophotometer (DU
640, Beckman, Fullerton, CA). Afterwards, 100 µl of AChE substrate
(42 mM acetylthiocholine) were added to the cuvette, and the change in
absorbance per minute was measured. Once the background absorbance was
subtracted, the AChE activity was calculated as international units
(IU) by means of the following equation
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where A is the change in absorbance per minute;
1.36 × 104 is the extinction coefficient of DTNB; Co
is the amount of tissue in the supernatant (mg tissue/ml buffer); 3,200 is the total volume (µl) of the cuvette; and 300 is the volume (µl)
of the supernatant sample.
Regarding blood samples, after 1:25 dilution in phosphate buffer, 100 µl were added to a cuvette containing 2.5 ml of DTNB and 500 µl
phosphate buffer. The remaining steps were the same as those described
for lung homogenates, with the corresponding change in the formula (100 µl of sample instead of 300 µl).
Drugs.
ACh chloride and His dihydrochloride (Sigma Chemical, St. Louis, MO)
were dissolved in saline solution. Acetylthiocholine and DTNB were
purchased from Aldrich Chemical (Milwaukee, WI) and were dissolved in
Tris-phosphate buffer at pH 7.38.
Statistical analysis.
For the assessment of airway responsiveness, the provocative agonist
dose that caused a 200% increment in RL and
iRL above baseline values (PD200) was
calculated by interpolation in a straight-line regression analysis.
Student's t-tests for unpaired data were used in most cases
and for paired data in those animals receiving two dose-response curves
sequentially. Statistical significance was set at two-tailed
P < 0.05. Data are expressed in the text and in Fig. 7
as means ± SE.
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RESULTS |
Basal values of pulmonary function and their relationship with
airway responsiveness.
Table 1 shows the baseline values from
every studied group, either as iRL or RL,
according to the plethysmographic method used. There were no
differences in most experimental vs. control pairwise comparisons,
except in three pairs of groups. Additionally, there was no correlation
between baseline iRL or RL and their corresponding PD200 value (data not shown).
SB/NEB (freely moving) group.
When the inhalatory route was used to measure airway responsiveness
through plethysmography for freely moving animals, the control group
exposed to air showed lower responses to the second ACh curve, i.e., a
clear hyporesponsiveness was developed (PD200: 0.41 ± 0.12 mg/ml, first curve vs. 0.86 ± 0.24 mg/ml, second curve; n = 6; P < 0.05; Fig.
1A). One-hour exposure to
O3 did not modify the airway responsiveness to ACh
(PD200: 0.50 ± 0.07 mg/ml before vs. 0.96 ± 0.24 mg/ml after O3; n = 6; Fig.
1B). However, when the duration of O3 exposure
lasted longer (3 h), hyperresponsiveness to ACh was observed
(PD200: 0.40 ± 0.06 mg/ml before vs. 0.27 ± 0.09 mg/ml after O3; n = 11;
P < 0.05; Fig. 1C).
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Fig. 1.
Effect of different stimuli on the airway responsiveness to
aerosolized ACh, expressed as the provocative dose 200%
(PD200), in spontaneously breathing/nebulized (freely
moving) guinea pigs. The first dose-response curve for ACh (open
circles) was performed 24 h before exposure to air (A),
ozone (B and C), or antigen (D),
whereas the second curve (shaded circles) was done in the same animal
16-18 h after air or ozone exposure or 3 h after the
antigenic (ovalbumin) challenge. ppm, Parts/million; [acetylcholine],
ACh concentration. Horizontal bars indicate the mean of the group.
Significant difference, * P < 0.05 and
** P < 0.0005.
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The PD200 to aerosolized His was not different before and
after exposure to air (PD200: 0.048 ± 0.01 vs.
0.052 ± 0.01 mg/ml; n = 7; Fig.
2) or before and after O3
exposure for 1 h (0.06 ± 0.02 vs. 0.05 ± 0.01 mg/ml;
n = 6).
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Fig. 2.
Effect of air (A) or ozone (B)
exposure on the airway responsiveness to aerosolized histamine,
expressed as the PD200, in spontaneously
breathing/nebulized (freely moving) guinea pigs. The first
dose-response curve for histamine (open circles) was performed 24 h before the exposure, whereas the second curve (shaded circles) was
done in the same animal 16-18 h after the exposure. [Histamine],
histamine concentration. Horizontal bars indicate the mean of the
group.
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Finally, airway hyperresponsiveness was developed in
ovalbumin-sensitized animals after an antigenic challenge, as
PD200 of nebulized ACh was statistically lower after such
challenge (0.24 ± 0.10 vs. 0.67 ± 0.12 mg/ml;
n = 5; P < 0.0005; Fig.
1D).
SB/NEB (restricted movements) group.
O3 exposure did not modify the ACh and His airway
responsiveness compared with their respective air-exposed groups. Thus
similar PD200 values were observed after air and
O3 exposure, either for the ACh challenge [0.68 ± 0.18 mg/ml (n = 6) vs. 0.56 ± 0.10 mg/ml (n = 4); P = 0.65; Fig.
3A] and the His challenge
[0.07 ± 0.01 mg/ml (n = 4) vs. 0.07 ± 0.01 mg/ml (n = 4); P = 0.94; Fig.
4A].
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Fig. 3.
Effect of air or ozone exposure on the airway
responsiveness to ACh, expressed as the PD200, in
spontaneously breathing/nebulized (A) and spontaneously
breathing/intravenous (restricted movements) (B) guinea
pigs. Each dose-response curve was done in separate animals 16-18
h after the exposure. Horizontal bars indicate the mean of the group.
Significant difference, * P < 0.01.
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Fig. 4.
Effect of air or ozone exposure on the airway
responsiveness to histamine, expressed as the PD200, in
spontaneously breathing/nebulized (A) and spontaneously
breathing/intravenous (restricted movements) (B) guinea
pigs. Each dose-response curve was done in separate animals 16-18
h after the exposure. Horizontal bars indicate the mean of the group.
Significant difference, * P < 0.005.
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SB/IV (restricted movements) group.
Contrasting with the SB/NEB (restricted movements) group, when ACh or
His were delivered intravenously, O3 generated airway hyperresponsiveness to both agonists with the shortest exposure (1 h)
(Figs. 3B and 4B). Thus ACh or His
PD200 values in guinea pigs submitted to O3
[7.36 ± 2.74 µg/kg (n = 6) and 1.41 ± 0.51 µg/kg (n = 6), respectively] were significantly
lower than in those animals just receiving air [25.14 ± 4.28 µg/kg (n = 7), P < 0.01; and
9.38 ± 1.78 µg/kg (n = 7), P < 0.005, respectively].
MV/NEB group.
O3 exposure did not change the airway responsiveness when
nebulized agonists were administered to mechanically ventilated animals, as ACh PD200 values (0.84 ± 0.19 mg/ml;
n = 6) were not statistically different from those
observed in the air-exposed group (1.61 ± 0.30 mg/ml;
n = 7; P = 0.062; Fig.
5A). Similarly, His
PD200 values between O3- and air-exposed groups
were similar [0.15 ± 0.06 mg/ml (n = 7) and
0.19 ± 0.07 mg/ml (n = 7), respectively; P = 0.70; Fig.
6A].
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Fig. 5.
Effect of air or ozone exposure on the airway
responsiveness to ACh, expressed as the PD200, in
mechanically ventilated/nebulized (A) and mechanically
ventilated/intravenous (B) guinea pigs. Each dose-response
curve was done in separate animals 16-18 h after the exposure.
Horizontal bars indicate the mean of the group. Significant difference,
* P < 0.05.
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Fig. 6.
Effect of air or ozone exposure on the airway
responsiveness to histamine, expressed as the PD200, in
mechanically ventilated/nebulized (A) and mechanically
ventilated/intravenous (B) guinea pigs. Each dose-response
curve was done in separate animals 16-18 h after the exposure.
Horizontal bars indicate the mean of the group. Significant difference,
* P < 0.02.
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MV/IV group.
By using anesthetized animals, exposure to 3 ppm O3 during
1 h generated airway hyperresponsiveness to the intravenous ACh and His. Thus after O3, ACh PD200 (10.26 ± 2.43 µg/kg; n = 6; Fig. 5B) was
significantly lower compared with the air group (50.38 ± 16.14 µg/kg; n = 6; P < 0.05). Similarly,
His PD200 was significantly lower for the
O3-exposed group than for air-exposed animals [8.85 ± 1.94 µg/kg (n = 6) vs. 23.39 ± 5.50 µg/kg
(n = 5), respectively; P < 0.02; Fig.
6B].
AChE activity.
O3 exposure did not cause statistical changes in the AChE
activity in either lung homogenates [1.022 ± 0.345 IU
(n = 6) vs. control, 0.301 ± 0.069 IU
(n = 5); P = 0.09] or total blood
samples [1.926 ± 0.470 IU (n = 6) vs. control,
1.320 ± 0.232 IU (n = 4); P = 0.35; Fig. 7].
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Fig. 7.
Effect of air or ozone exposure on the
acetylcholinesterase activity in lung homogenates (A) or
blood (B). International units (IU) correspond to moles of
substrate hydrolyzed per minute per gram of lung tissue, or to moles of
substrate hydrolyzed per minute per milliliter of blood. Values are
means ± SE.
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DISCUSSION |
O3-induced airway hyperresponsiveness is a
well-characterized phenomenon widely studied in laboratory animals
since many years ago. Nevertheless, several factors might influence the
resulting effects of O3, such as concentration and duration
of the exposure, time elapsed between exposure and bronchoprovocation
tests, and nature of the bronchoconstrictor agent used to demonstrate
the hyperresponsiveness. An additional factor that has been very
scantily studied is the administration route utilized to deliver the
bronchoconstrictor agonist (18). In the present study, we
found that, at least under our experimental conditions (3 ppm
O3 during 1 h), bronchoprovocation tests performed
with nebulized agents were unable to demonstrate the
O3-induced airway hyperresponsiveness (as can be seen in
Figs. 1B, 2, 3A, 4A, 5A,
and 6A), whereas intravenously delivered agents always
revealed it (as observed in Figs. 3B, 4B,
5B, and 6B). These contrasting responses
were independent of the plethysmographic method used.
Our results differ from some previously published works made in guinea
pigs, in which airway hyperresponsiveness to inhaled agonist was found
shortly after a 3 ppm O3 exposure during 1 h or less
(10, 14, 17, 23). Most of these studies, however, measured
the airway reactivity when a significant change in baseline airway
caliber was present (between 60 and 120% increment), and thus such
hyperresponsiveness could be explained solely due to mechanical
reasons. In this sense, we have found that this transient bronchoconstriction might last for up to 3 h (21),
and thus O3-induced airway hyperresponsiveness due to
nonmechanical processes should be measured once this obstructing phase
has ended. Although we took care to avoid large O3-induced
changes in the basal values of iRL and RL, we
found a statistically significant increase in two groups. In both
cases, the increment was <19%, and an associated hyperresponsiveness
was observed in only one of them. Moreover, there was no correlation
between iRL or RL basal values and their corresponding airway responsiveness, either in individual groups or in
pooled SB/NEB freely moving animals (data not shown). Thus it is
probable that the O3-induced changes in airway
responsiveness obtained in our study were not due to modifications in
basal values.
Contrasting with our results observed with 3 ppm O3
exposure during 1 h, we observed that a longer exposure time (3 h)
was capable of producing airway hyperresponsiveness to inhaled agonists (Fig. 1, B and C, respectively). In this sense,
some researchers have exposed guinea pigs to the same O3
concentration for up to 2 h and found airway hyperresponsiveness
at 5 h (17), or at 4, 14, and 24 h
(18) after the exposure. Therefore, at least at 3 ppm,
duration of the exposure to O3 seems to influence the development of airway hyperresponsiveness to inhaled agonists.
In general, our results strongly suggest that the administration route
was the main factor responsible for the characterization of the
O3-induced airway hyperresponsiveness. This is in agreement with a previous report by Roum and Murlas (18), in which
they concluded that the inhalatory technique was not appropriate to demonstrate O3-induced airway hyperresponsiveness to
cholinergic agonist because of its great variability, whereas the
intravenous method was more consistent. The disparate responses
observed when bronchoconstrictors were delivered via aerosols or
intravenously might have several explanations.
First, previous studies have shown that O3 exposure causes
morphological (4, 13, 19) and functional (3)
changes in the peripheral airways. Thus, if peripheral airways are the site at which O3 is causing the major functional
disturbances, one possibility is that inhaled bronchoconstrictor
agonists did not properly reach such distal regions. The intravenous
route, by contrast, should reach all of these locations homogeneously, thus detecting the O3-induced dysfunction. A different site
of action of inhaled and intravenous agonists can also be inferred from
the study by Corddry et al. (8). They found that
responsiveness measured through whole RL and peripheral Raw
closely matched during an intravenous bronchial challenge to His,
whereas both measurements yielded different results with nebulized His.
This argument might also be reasonable to interpret the results
obtained by increasing the time of O3 exposure to 3 h
(Fig. 1C), because this stronger stimulus could be producing
dysfunction at a more proximal airway level. This postulate would also
explain the airway hyperresponsiveness to aerosolized agents after
antigen challenge in sensitized guinea pigs observed by others
(5) and us (Fig. 1D), because it is well known
that sensitization in animal models occurs at all airway levels.
Second, an increased arrival of the intravenously administered agonists
to the airway smooth muscle would also explain the different effect of
O3 on airway hyperresponsiveness. In this sense, it is
known that O3 exposure produces an increased vascular permeability (16), which might favor an augmented delivery
of the agonists to the airway smooth muscle that would not occur when
the agonists are delivered by the inhalatory route. Further research is
needed to assess the certainty of this hypothesis. On the other hand,
some studies have found that tissue and blood AChE activity diminishes
shortly after O3 exposure (2, 11). Thus it is
reasonable to speculate that, in our study, the inhibition of AChE in
lung tissue was not enough to produce airway hyperresponsiveness to
inhaled ACh, but, when the intravenous route was used, the additional
AChE inhibition in blood would cause more ACh to reach the airway
smooth muscle, thus causing airway hyperresponsiveness. However, we
found that, under our experimental conditions, O3 did not
inhibit AChE activity (in fact, an opposite trend was noticed; Fig. 7).
This discrepancy with published works might be explained by the longer
lapse between the O3 exposure and the AChE activity
measurement in our work. In fact, in preliminary experiments, we have
corroborated that an acute O3 exposure (3 ppm, 1 h)
causes a transient diminution of blood AChE activity in rabbits, which
returned to control levels at 16-18 h after exposure (data not
shown). Thus AChE inhibition seems not to be involved in the
differential effects of inhaled and intravenous agonists to demonstrate
O3-induced airway hyperresponsiveness.
Finally, as can be seen in Fig. 1A, in the air-exposed
SB/NEB freely moving animals, we found that a second administration of
aerosolized ACh was notably less efficacious than the first one, i.e.,
a hyporesponsiveness to this agonist was developed. This phenomenon did
not occur when His was nebulized nor when either agonist was
administered by the intravenous route (data not shown). The nature of
this ACh-induced hyporesponsiveness is unclear. It might be possible
that aerosolized ACh activated prejunctional M2 receptors
(with a consequent inhibition of basal ACh release) or promoted the
production of a relaxant factor more effectively than intravenous ACh.
Nonetheless, the role of this phenomenon, if any, in the lack of
O3-induced hyperresponsiveness to inhaled agonists remains
speculative and deserves further investigation.
In conclusion, in the two plethysmographic methods used by us, we found
that inhaled agonists were less effective to reveal the airway
hyperresponsiveness after an acute O3 exposure than intravenous ones, at least for the 1-h exposure to 3 ppm. This difference seems not to be related to an O3-induced
inhibition of the AChE activity.
| |
ACKNOWLEDGEMENTS |
Consejo Nacional de Ciencia y Tecnologia and Instituto Mexicano del
Seguro Social provided scholarships supporting postgraduate studies in
which this work was done (to B. Sommer).
| |
FOOTNOTES |
*
L. M. Montaño and M. H. Vargas contributed
equally to this work.
Address for reprint requests and other correspondence: L. M. Montaño, Departamento de Investigación en Asma, Instituto
Nacional de Enfermedades Respiratorias, Tlalpan 4502, CP 14080, México DF, México (E-mail: lmmr{at}servidor.unam.mx).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 December 2000; accepted in final form 15 August 2001.
| |
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