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Meakins-Christie Laboratories, Royal Victoria Hospital, Montreal General Hospital, and Montreal Chest Institute Research Center, Montreal, Quebec, Canada H2X 2P2
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Wang, C. G., J. J. Almirall, C. S. Dolman, R. J. Dandurand,
and D. H. Eidelman. In vitro bronchial responsiveness in two
highly inbred rat strains. J. Appl.
Physiol. 82(5): 1445-1452, 1997.
We investigated
methacholine (MCh)-induced bronchoconstriction in explanted airways
from Fischer and Lewis rats. Lung explants, 0.5- to 1.0-mm thick, were
prepared from agarose-inflated lungs of anesthetized 8- to 12-wk-old
male rats. After overnight culture, videomicroscopy was used to record
baseline images of the individual airways. Dose-response curves to MCh
were then constructed by repeated administration of MCh; airways were
reimaged 10 min after each MCh administration. Airway internal luminal
area
(Ai)
was measured at successive MCh concentrations from
10
9 to
101 M. In
addition to the effective concentration leading to 50% of the achieved
maximal response, we also determined the effective concentration
leading to a 40% reduction in
Ai.
Both the effective concentration leading to 50% of the achieved
maximal response and the concentration leading to a 40% reduction in
Ai
were significantly lower among Fischer rat airways
(P < 0.05). Airway closure was more
common among Fischer rat airways (17%) than among those of Lewis rats
(7.5%). Responsiveness of Fischer rat airways was more heterogeneous
than among Lewis airways; a larger number of Fischer rat airways
exhibited high sensitivity to MCh. There was no relationship between
responsiveness and baseline
Ai
in either strain. In a second experiment, we measured the rate of
contraction of explanted airways from lungs inflated to 50, 75, and
100% of total lung capacity. The average rate of contraction in the
first 15 s was higher in Fischer rat airways at each inflation volume.
These data indicate that the hyperresponsiveness of the Fischer rat reflects the responsiveness of individual airways throughout the airway
tree and are consistent with the notion that in this model hyperresponsiveness is an intrinsic property of airway smooth muscle.
heterogeneity; Fischer rat; Lewis rat
INTRODUCTION INBRED RAT STRAINS exhibit characteristic degrees of
bronchial responsiveness when challenged with airway smooth muscle
agonists in vivo (18). In particular, the Fischer 344 rat has been
found to be significantly more responsive to methacholine (MCh) than are animals of the Lewis strain (6, 8, 10, 18). This relative
hyperresponsiveness is manifested as a shift in the dose-response curve
to aerosolized MCh in both spontaneously breathing (10) and
mechanically ventilated animals (6). Similar interstrain differences
have been found in the mouse (9, 16), suggesting that this is not a
species-specific phenomenon and that these models may provide clues
regarding the genetic determinants of responsiveness in humans.
A number of phenotypic differences between Fischer and Lewis strains
that may contribute to differences in bronchial responsiveness have
been observed. Fischer rats have more airway smooth muscle (10) and
appear to have a lower degree of airway-parenchymal interdependence
compared with the Lewis strain (6). There is no correlation within each
strain between airway smooth muscle quantity and responsiveness (10),
nor does there appear to be a difference in the in vivo plateau
response between Fischer and Lewis rats (6). Both of these observations
are against the notion that smooth muscle bulk alone explains the
hyperresponsiveness of the Fischer rat. Fischer rat airway smooth
muscle shows a greater capacity to proliferate in vitro than does Lewis
rat muscle (23), and this is associated with increased in vitro
isometric force generation (12). Furthermore, airways from Fischer rats
may have a lower capacity to relax in response to nitric oxide compared with those of Lewis rats (12). None of these differences
appear to completely explain the relative hyperresponsiveness of the Fischer rat, but taken together they appear to localize mechanism to
the airways themselves, probably at the level of the airway smooth
muscle. The present study was, therefore, aimed at defining the
responsiveness of individual airways from both Fischer and Lewis rats.
We have previously found evidence of extensive parallel heterogeneity
of responsiveness in the rat by using cultured lung explants (5).
Airways of similar dimension from different locations within the lung
exhibited a wide variation in responsiveness to MCh. This phenomenon
does not appear to be restricted to the rat because similar
observations have been made in the dog (4, 17). It is unclear, however,
how this heterogeneity relates to responsiveness of the lung as a
whole. We specifically wished to determine whether differences between
Fischer and Lewis strains could still be detected despite the
anticipated variability in responsiveness of individual airways. To do
this, we compared airways from Fischer and Lewis rats by using the
approach of Dandurand and co-workers (5), which allows construction of
individual dose-response curves in several airways from the same animal
so that an estimate of the intraindividual variation in responsiveness may be made. We reasoned that if the locus of hyperresponsiveness were
the bronchial smooth muscle, then Fischer rat airways should be
hyperresponsive throughout the airway tree so that hyperresponsiveness would be detectable regardless of heterogeneity.
A second goal of this study was to investigate whether differences in
responsiveness among inbred rat stains extended to contractile events
immediately after stimulation. It is during this time period that most
airway smooth muscle shortening occurs, corresponding to rapid
cross-bridge cycling (20). To the extent that differences in bronchial
responsiveness among inbred strains are related to the contractile
properties of airway smooth muscle, these differences should be
detectable very early in contraction. Specifically, the rate of
contraction of airways from Fischer rats should be higher than that of
Lewis rats. To investigate this possibility, we extended the explant
technique to permit measurements of the dynamics of bronchoconstriction
at the beginning of the contraction by recording the changes in
internal luminal area of airways
(Ai) after MCh stimulation.
METHODS
-2-ethanesulfonic
acid (HEPES)-buffered culture medium (HCM) was prepared in a manner
identical to the BCM preparation except that 5.96 g/l HEPES
(Sigma) were substituted for sodium bicarbonate and the pH adjusted to
7.25.
Agarose solutions.
Agarose (2.0 and 4.0%; type VII, Sigma) solutions were prepared with
BCM without supplements. The agarose solutions were autoclaved, then
were stored at 4°C, and were available for use within 1 wk.
Preparation of explants.
Animals were anesthetized with a lethal dose of pentobarbital sodium
(80 mg/kg ip) and were placed supine in a laminar flow hood.
Sterilization of the external surfaces of the animal was performed with
70% ethanol. A midline incision was made from the neck to the abdomen,
and the trachea was exposed. Tracheostomy and intubation were performed
with sterile polyethylene tubing (PE-240) 6 cm in length. The anterior
wall of the abdomen was opened, and the animal was exsanguinated by
inferior vena cava section. The anterior chest wall was then removed,
allowing the heart, lungs, and trachea to be removed en bloc and placed
in a sterilized plastic cup with a hole in the lid to permit the tracheal tube to protrude.
Agarose (2%) was melted in a microwave oven, and then the liquid
agarose was cooled to 37°C for use. Equal volumes of the liquid 2%
agarose solution and BCM containing double supplements warmed to
37°C were mixed, resulting in a 1% agarose-BCM solution. The
excised lungs were slowly inflated with the warmed 1% agarose-BCM solution to an inflation volume equal to the desired percentage of
predicted total lung capacity (TLC) based on the animal's weight (TLC = 48 ml/kg) (6). This was followed by injection of a 1.0-ml bolus of
air to clear the conducting airways of agarose. The inflation volume
was then maintained by clamping the tracheal tube. The preparation then
was cooled to 4°C for 30 min to allow the liquid agarose-BCM
solution in the lungs and airways to gel. The lungs were then dissected
away from the heart and placed upright in a sterile syringe cylinder,
the needle end of which had been cut off. Liquid agarose (4%) at
37°C was poured into the cylinder to embed the isolated lungs, the
top end opening was plugged with a rubber stopcock, and then the
agarose was allowed to gel at 4°C for 30 min.
The lung-agarose block was sectioned into 0.5- to 1.0-mm transverse
slices by using a handheld microtome blade (model 818, Cambridge
Instruments, Buffalo, NY). The slices were inspected at ×10
magnification by using an inverted microscope (Olympus OMT-2, Tokyo,
Japan), and those that included at least subsegmental and smaller
airways were each placed in a 30-mm culture well insert (Millipore,
Bedford, MA) within a six-well culture plate (Costar, Cambridge, MA),
containing 2 ml of BCM in each well. The lung explants were incubated
overnight at 37°C with 5%
CO2-95% air ventilation.
Protocol 1: Concentration-response curves.
The culture dish inserts containing the lung explants were transferred
to another six-well plate (Costar) with 2 ml of the HCM in each well
and placed on the temperature-controlled stage of the inverted
microscope that was equipped with a video camera (model CCD-200-R,
Videoscope, Washington, DC). All studies were carried out at 37°C.
Each explant was scanned to locate an airway for study on that slice.
Airways were deemed suitable for study if they were cut in cross
section (long axis-to-short axis ratio <2:1) and the epithelium was
free of agarose. After baseline images of selected airways were
recorded with a videodisk recorder (Panasonic TQ 2026F, Osaka, Japan),
a 20-liter aliquot of stock MCh-HCM solution was added to each of the
wells outside the culture inserts, resulting in a final MCh
concentration of 109 M. After incubation at 37°C for 10 min, all the airways were reimaged.
This procedure was repeated for each concentration of MCh. Initial
experiments were conducted by using a maximal concentration of
102 M. This was increased
to 101 M when few plateau
responses were observed among the first six Lewis rats studied. All
Fischer rats appeared to reach a plateau response by
102 M.
Construction of concentration-response curves for each airway was
performed by plotting
Ai
as a percentage of baseline value against the MCh concentrations (from
109 to
102 or
101 M). The plateau of a
concentration-response curve was defined when the last two or three
points on the curve differed by <10% of baseline value or the airway
reached at least 90% of airway closure after the highest MCh
concentration. Maximal response (MR) was defined as the minimal area
obtained from an airway but expressed as a percentage of complete
closure of the airway; i.e., MR = [1 
(minimal
Ai/baseline
Ai) × 100].
Airways with maximal changes in
Ai
<25% of baseline values were excluded to avoid inadvertent
measurement of pulmonary vessels and nonspecific airway smooth muscle
shortening (5). As a measure of bronchial responsiveness, we calculated
EC50 as the effective concentration causing a reduction in
Ai
equal to 50% of the MR. EC50 was
determined by linear interpolation on log-transformed MCh
concentrations, and it is expressed in log units. We found, however,
many airways from Lewis rats did not reach a plateau even at
101 M. We, therefore, also
calculated another index of reactivity (ECA40)
as the effective concentration causing a 40% decrease in
Ai
from the baseline value, and it is expressed in log units.
ECA40
is independent of the maximal response.
Protocol 2: Dynamic responses
The protocol was aimed at determining the dynamic response behavior of
airway smooth muscle to MCh. We studied explants from lungs inflated to
three different volumes: 100% TLC (7 Fischer and 8 Lewis rats), 75%
TLC (6 Fischer and 8 Lewis rats), and 50% TLC (5 Fischer and 5 Lewis
rats). Culture dishes containing the lung explants were transferred to
another six-well plate with 2 ml of HCM in each well and placed on the
stage of the inverted microscope. Explants for
protocol 2 were screened for the
presence of airways and selected for study by using the same approach
as in protocol 1. After the baseline
image was recorded, 20 ml of 102 M MCh solution were
dropped directly on the explant tissue (final concentration
104 M), and the airway
images were simultaneously taken and sequentially recorded at
frequencies of 1 Hz for 15 s and changed to 0.2 for the next 15 s,
0.033 Hz for the next 30 s, and then 0.016 Hz for the remainder of the
5-min recording.
Dynamic responses were measured in explants from lungs inflated to 50 (5 Fischer and 5 Lewis rats), 75 (6 Fischer and 8 Lewis rats), and
100% (7 Fischer and 8 Lewis rats) of TLC. As can be seen in Table
1, there was a significant effect of
inflation volume on baseline
Ai
[P < 0.05, 2-way
(ANOVA)], although there was no significant difference in
Ai
between the two strains at any lung volume or overall. In
this protocol, MR was calculated as the largest change from baseline
over 300 s. Rate of contraction was evaluated by using two indexes
based on the internal perimeter (Pi),
which was calculated as 2
by assuming a circular lumen so that
r = (Ai/)1/2,
where r is radius. We
calculated the average velocity of shortening during the first one-half
of the constriction as the change in Pi
from baseline to 50% of the maximal achieved contraction divided by
the elapsed time
(
Pi/t).
We also calculated the peak velocity by numerically differentiating the
values of
Pi
with respect to time by using
(dPi/dt)
and choosing the maximal value. To control for
differences in baseline smooth muscle length, all measurements of
velocity were normalized for baseline
Pi.
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RESULTS
3 M. The variability in
responsiveness among rats was much less than that of airways within
each given individual, which spanned several log doses in some cases.
Although there was overlap between the two strains, airways from
Fischer rats tended to be more responsive, with more airways exhibiting
EC50 values below 10 3 M. Fischer rat airways
also showed a higher prevalence of airway closure (17%) than did
airways from Lewis rats (7.5%) in this protocol
(P < 0.05).
Figure 2 shows the frequency distributions of Fischer and Lewis rat airways after the data were pooled by rat strain, allowing the differences between strains to be seen more clearly. Statistical analysis of these curves reveals several significant differences between Fischer and Lewis rats (Table 2). The geometric means of EC50 and ECA40 for the Fischer group were
3.57 and 3.48, significantly
lower than those for the Lewis group (2.81 and 2.59,
respectively; P < 0.05). Similar
results were found for the median values (Table 2). The distributions
were slightly asymmetrical with a notable tail of highly responsive
airways, particularly in the Fischer strain. Consistent with this,
although we found no significant difference in distribution symmetry
(skewness), kurtosis among Fischer airways was significantly less than
among Lewis airways (P < 0.003). The
lower kurtosis value reflects a relatively large left-hand tail of the
distribution (Fig. 3), which is accounted
for by a larger number of highly responsive airways among the Fischer
animals. This heterogeneity is also reflected in the IQD, which tended
to be greater among Fischer than Lewis animals (Table 2).
, Fischer rats; 
, Lewis rats. Each point represents
value for an individual airway. Distributions of responsiveness is
different between the 2 strains with either index.
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Protocol 2. Typical examples of the dynamic response to MCh are shown in Fig. 4. It can be seen that the peak in dPi/dt occurs ~2 s after drug administration. Most of the smooth muscle shortening occurs within the first 30 s, with one-half of the contraction complete before 10 s. As in the concentration-response protocol, the MR to MCh was greater among Fischer compared with Lewis rat airways (Fig. 5). This was true at all three inflation volumes: 50% TLC, 86.1 ± 3.6 vs. 79.1 ± 3.6; 75% TLC, 81.2 ± 2.3 vs 76.7 ± 2.8; 100% TLC, 78.2 ± 3.5 vs. 67.7 ± 4.1 (P < 0.001; Fig. 5A). The average rate of contraction over the first one-half of contraction (
Pi/t)
was also higher among Fischer than Lewis rats at all lung volumes: 50%
TLC, 7.0 ± 1.1 vs. 5.8 ± 0.6; 75% TLC, 8.0 ± 0.5 vs. 5.6 ± 0.4; 100% TLC, 4.8 ± 0.5 vs. 4.1 ± 0.5 (P < 0.001; Fig.
5B). Peak
dPi/dt
also tended to be higher at all inflation volumes (Fig.
6A),
although this reached statistical significance only in the 75% TLC
group. There was no difference between strains with regard to the time
to peak velocity. (Fig. 6B).
4 M MCh at 3 different
inflation values. B: average rate of
contraction over first 15 s in same airways. Both maximal rate and rate
of contraction are higher among Fischer compared with Lewis rats regardless of degree of inflation. TLC, total lung capacity.
* Statistically significant difference between the 2 strains,
P < 0.05.
4 M MCh at 3 different inflation values. B: time to
peak value for the same airways. Although there was a tendency for peak
dPi/dt to be higher in Fischer rat airways, this did not reach statistical significance. No difference in time to peak was observed.
DISCUSSION
We measured the responsiveness of individual intraparenchymal airways in Fischer and Lewis rats and confirmed previous observations indicating differences between those two rat strains (6, 10). Fischer rats were more reactive by almost every parameter studied. Sensitivity to MCh, MR, and the dPi/dt were higher among Fischer rats, which also appeared to exhibit a higher degree of heterogeneity of responsiveness within the airway tree.
We found an interstrain difference in MCh responsiveness of approximately one log dose between Fischer and Lewis rats in this study regardless of the method used to calculate responsiveness (i.e., EC50 or ECA40). This difference is approximately twice as large as that observed in vivo, in which Fischer rats have usually been found to be more responsive than Lewis rats by about two doubling doses (i.e., a factor of 4) (6, 10). The larger in vitro interstrain separation may have arisen for several reasons. First, the explant method provides more detailed information about each animal than do the in vivo techniques. Additionally, the present studies more directly reflect airway smooth muscle function and are free of confounding information related to drug delivery, ventilatory regime, and so on. The improved separation between rat strains is good evidence that the relative hyperresponsiveness of the Fischer rat occurs at the level of the airway itself.
A key advantage of using lung explants was the possibility of studying airways from many parts of the intraparenchymal airway tree. Previous in vitro studies of this model have focused on the trachea. Tracheal smooth muscle from Fischer rats has been reported to be hyperresponsive compared with that from Lewis rats (8, 12). Our present observations indicate that differences between Fischer and Lewis rats are not restricted to the trachea but extend throughout the airway tree. It is unclear whether the same situation pertains in humans. Bai (1, 2) has reported that airway smooth muscle from trachea, but not bronchi, of patients dying of asthma may be hyperresponsive in vitro. Although these studies are not directly comparable to ours, the observation of hyperresponsive peripheral airways in the rat may represent a different mechanism from that seen in humans.
Fischer rat airways exhibited a greater capacity for airway narrowing both in terms of airway closure detected in protocol 1 and in magnitude of the MR at a constant MCh dose in protocol 2. It is noteworthy that only a minority of airways achieved closure in the protocol 1. This may have been a consequence of the experimental conditions, however. Dandurand and co-workers (5) previously reported that airway closure in explanted airways is sensitive to both agarose concentration and inflation volume. We used a high inflation volume for the concentration-response curves to maximize image quality, and this may have minimized the number of closed airways observed. Nevertheless, in protocol 1, maximal contraction was clearly greater among airways from Fischer rats compared with those from Lewis rats regardless of inflation volume. Airways from Fischer rats clearly have a greater capacity to close than do those from the Lewis strain.
In addition to differences in concentration-response curve position and MR, we found evidence that the distribution of Fischer rat airways differed from that of Lewis rats (Fig. 3, Table 2). Kurtosis, a measure of the size of the tails of the frequency distribution, was significantly greater among Fischer rat airways, as accounted for by a number of very responsive airways. On the other hand, the SD and the skewness were similar in the two strains. Thus, in terms of the most responsive airways, Fischer rats are more heterogeneous than Lewis rats, although the width of the distributions near the mean (expressed by the SD) is similar in the two strains. It is difficult to know what the physiological implications of this difference in frequency distributions are. Bates (3) has recently pointed out that a stochastic distribution of airway properties has the potential to profoundly influence measurements of resistance and by implication responsiveness (3). Using Monte-Carlo simulation of a constricting airway tree, he found that stochastic variation of the structural characteristics of individual airways on its own resulted in relatively large degrees of variability in measured resistance (3). This arises at least in part because of the highly nonlinear relationship between radius and airway resistance (3). Bates, however, used the SD as his measure of stochastic variability, so it is difficult to directly apply his analysis to the present data in which differences in kurtosis were found. Furthermore, we measured functional parameters of responsiveness rather than structural properties. Nevertheless, the modeling results suggest that the nature of the distribution of bronchial responsiveness across the airway tree needs to be considered as a potential contributing factor in the differences between Fischer and Lewis rats measured in vivo.
We extended the explant technique to look at the dynamics of the response to MCh stimulation in explants by measuring the rate of change of the Pi of the airways after MCh administration. There are several limitations to this approach that must be taken into account in interpreting the data. First, the explant preparation cannot be used to directly provide an estimate of the maximal isotonic shortening rate because this would require zero-load conditions (20). In the present case, there is no convenient way to precisely ascertain either preload or afterload, so we tried to account for the effect of load by making the measurements at different lung volumes. Another problem is that measurement of the change in Pi is an indirect index of airway smooth muscle shortening for at least two reasons. First, airway smooth muscle is not precisely circumferential in its arrangement; recent measurements in cat, human, and guinea pig airways suggest a slightly oblique orientation near 13° (15, 19). Second, particularly at high degrees of constriction, folding of the airway epithelium is likely to occur. In addition to potentially presenting a mechanical load to airway smooth muscle shortening (14), very high degrees of folding would be missed in our measurements because the separations between epithelial folds are not always evident on videomicroscopic images. On the other hand, because our measurements of contraction rate focused on the earliest part of the constriction, it is unlikely that epithelial folding resulted in a large error.
Despite these limitations, the dynamic data are reminiscent of published measurements of the velocity of shortening of airway smooth muscle. We observed that dPi/dt peaked rapidly and declined quickly with time so that one-half of the contraction was completed 7-8 s after onset (Fig. 4). When measured by using the zero-load clamp technique under isotonic conditions, the velocity of shortening of canine tracheal smooth muscle after electrical field stimulation peaks within 2 s and declines to 70% of maximum within 10 s. Seventy-five percent of the maximum shortening capacity is completed within 2-2.5 s In contrast, under isometric conditions, velocity falls off only by ~30% over the course of the contraction (20). The airways in our system were clearly not under isotonic conditions; rather they constricted against a variable elastic load. Stephens and co-workers (20) have postulated that under in vivo auxotonic conditions, the velocity of shortening must lie between the extremes of isotonic and isometric conditions. In the present case, the dynamics of contraction more closely resembled results expected under isotonic than isometric conditions in that the rate of constriction fell off rapidly with time. To the extent that explants reflect the situation in vivo, bronchial smooth muscle shortening in the rat may be quasi-isotonic at least during the initial phase of contraction.
Using indexes of the dynamics of the contractile response in the first
few seconds after agonist administration, we found evidence that the
rate of contraction of Fischer rat airways was higher than that in
Lewis airways. The
Pi/t
was greater among Fischer airways regardless of the inflation volume
(Fig. 5B). There was also a trend
toward higher peak
dPi/dt,
but this reached statistical significance only at an inflation volume
of 75% TLC. These differences in the dynamics of contraction between
Fischer and Lewis strains may have implications for the underlying
mechanisms of hyperresponsiveness in this model. It has been proposed
that the shortening of airway smooth muscle immediately after
stimulation is accounted for by the activity of normally cycling cross
bridges (20). With sustained stimulation, shortening becomes dominated by more slowly cycling "latch bridges" (7, 13). Our dynamic data,
therefore, suggest that at least some of the phenotypic differences
between these strains are related to events involved in the early
activation of smooth muscle contraction such as second-messenger generation, mobilization of intracellular calcium, and the rate of
cross-bridge cycling. A recent report of more rapid development of a
rise of intracellular calcium in serotonin- and bradykinin-stimulated cultured Fischer rat smooth muscle cells is consistent with this notion
(21). In this regard, however, it is important to note that we did not
detect any difference in the time to peak
dPi/dt (Fig. 6B) as might be expected if
the differences in the rate of cellular activation were present.
Nevertheless, at the very least, our observation of differences in
dynamics supports the notion that the hyperresponsiveness in the
Fischer model is a function of the airway smooth muscle itself.
Although our in vitro measurements of responsiveness generally are consistent with previous findings in intact animals, the situation for MRs is less clear. While MRs were larger in Fischer rat airways in both protocols of this study, we previously failed to find a difference between Fischer and Lewis rats in the MR of mechanically ventilated rats in vivo (6). One possible explanation for this discrepancy is that differences in the MR were missed in vivo because of technical problems. End-tidal volume was not well controlled in our previous study, and this may have prevented us from accurately measuring MRs (6). It is also possible that the discrepancy between in vivo and in vitro findings reflects physiological differences. For example, it is possible that tidal oscillations reduced the contractility of the airways in vivo sufficiently to blunt the MR. Gunst et al. (11) have reported that tidal oscillation has the potential to greatly influence the responsiveness of airway smooth muscle, and Warner and Gunst (22) have suggested that this may play an important role in the intact lung. It is also possible that when resistance is measured in an intact animal, the effects of a higher MR in a subset of airways cannot be detected in the overall lung resistance. Further experimental and modeling studies are required to address this possibility.
In summary, we found the airways of Fischer rats to be hyperresponsive compared with those of Lewis rats by almost all the criteria we measured. This hyperresponsiveness appeared to be a property of airways of all sizes and was reflected even in contractile events occurring very soon after drug delivery. These findings strongly support the hypothesis that bronchial hyperresponsiveness in this model is a function of the intrinsic contractile properties of the airway smooth muscle.
ACKNOWLEDGEMENTS
We are indebted to Dr. Heberto Ghezzo for assistance with the statistical analysis. We thank Dr. M. Ludwig for reviewing the manuscript.
FOOTNOTES
Address for reprint requests: D. Eidelman, Meakins-Christie Laboratories, McGill Univ., 3626 St. Urbain St., Montreal, PQ, Canada H2X 2P2.
Received 3 July 1996; accepted in final form 23 December 1996.
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