Combined effect of chronic hypoxia and in vitro exposure to gas pollutants on airway reactivity

Etienne Roux1, Michel Duvert2, and Roger Marthan1

1 Laboratoire de Physiologie Cellulaire Respiratoire, Institut National de la Santé et de la Recherche Médicale Equipe Mixte 9937; 2 Laboratoire de Cytologie, Institut National de la Santé et de la Recherche Médicale Equipe Mixte 9929 and Institut Fédératif de Recherche n°4, Université Victor Segalen Bordeaux 2, 33076 Bordeaux cedex, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study investigated the interaction between exposure to air pollutants and chronic hypoxia (CH). We used a hypobaric chamber (14 days at barometric pressure 380 mmHg) to produce CH in rats. Exposure to various doses of acrolein or ozone did not modify the mechanical response to cholinergic agonists. Exposure to 3 µM/min acrolein did not alter epithelium-free trachea responsiveness. In contrast, direct exposure of freshly isolated myocytes to 2 and 3 µM/min acrolein enhanced the amplitude of the first intracellular [Ca2+] rise in response to 0.1 µM ACh and the calcium oscillation frequency in response to 10 µM ACh. CH alone did not alter smooth muscle cross-sectional area (SMA) or epithelium-plus-submucosa thickness. CH decreased maximal contractile response (maximal force normalized to SMA) but increased sensitivity (pEC50) to cholinergic agonists. We conclude that unlike in normoxic rats, exposure to air pollutants does not induce airway hyperresponsiveness in CH rats, although it increased calcium signaling. These results cannot be explained by change in smooth muscle accessibility, but may be linked to the effect of CH on calcium-contraction coupling.

ozone; acrolein; smooth muscle; rat trachea; excitation-contraction coupling


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INHALATION OF AIR POLLUTANTS induces airway hyperresponsiveness and, therefore, poses a possible risk to human health, especially in patients whose airways are already compromised by preexisting pathologies such as asthma or chronic obstructive pulmonary disease (COPD) (8, 9, 11). Unlike the interaction between asthma and air pollution that has largely been investigated by epidemiological, clinical, as well as experimental studies (16, 17, 29), very little is known about the combined effect of air pollution and chronic hypoxia (CH), a condition that is frequently observed in patients suffering from COPD (21, 31). The effect of CH, per se, on airway responsiveness remains unclear, since very few experimental studies have addressed this issue (1, 6).

We previously reported that ex vivo administration of a variety of gas pollutants, such as ozone or acrolein, to either human lung or rat isolated airways alters the subsequent in vitro mechanical response (2-4, 26, 29). This alteration is related to changes in calcium signaling at the site of the smooth muscle cell (15, 28). We have also developed an experimental model of CH using a hypobaric chamber and observed that CH modifies the mechanical response of isolated airways as a consequence of an effect on calcium signaling (1, 5).

The aim of the present study was thus to investigate the interaction between CH and exposure to pollutants in rat isolated trachea. We examined the effect in CH rats of preexposure to ozone and acrolein on the contractile response to muscarinic stimulation in isolated tracheal rings and that of preexposure to acrolein on calcium signaling in isolated myocytes. Experimental procedures and doses for exposure to air pollutants were chosen on the basis of previous dosimetric studies (1, 2, 26, 28), to allow valuable comparison between the results of the present study and those obtained in normoxic animals. We also examined the effect of CH on histological structure and contractile responsiveness of rat trachea.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CH

Male Wistar rats, 8- to 10-wk old, were exposed to a simulated altitude of 5,500 m (barometric pressure 380 mmHg) in a well-ventilated, temperature-controlled hypobaric chamber for 14 days as previously described (1, 5). Such a protocol generates CH, which in turn provokes pulmonary artery hypertension and right ventricular hypertrophy, shown by an increase in the ratio of right ventricle-to-left ventricle-plus-septum weight (RV/LVS) (1, 6, 20, 22). We measured, in a subset of the present animal population, the parameter RV/LVS (0.51 ± 0.02, n = 6) and found it increased consistently with previous studies (1, 22, 30). Free access to a standard rat diet and water was allowed throughout the exposure period. Normoxic rats were kept under similar conditions but not in the hypobaric chamber. Rats were weighed at the end of the 2-wk stay. We verified that 14 days of hypoxia had only a small and nonsignificant effect on rat weight [339 ± 5.7 and 319 ± 5.6 g for normoxic (n = 7) and CH (n = 20) groups, respectively].

Tissue Preparation

For each experiment, a rat was killed by cervical dislocation. The heart and lungs were removed en bloc, and the trachea was rapidly dissected out. For isometric contraction experiments, each trachea was cut into four rings of similar size, i.e., ~4 mm in diameter and 4 mm in length, verified under binocular control. For experiments performed in epithelium-free tracheal rings, we mechanically removed the epithelium by rubbing the lumen of the rings with a cotton-tipped applicator. The absence of epithelium as well as the integrity of the submucosa were verified by histological examination at the end of the experiments (not shown).

For fluorescence measurements of intracellular Ca2+ concentration ([Ca2+]i) in isolated cells, the muscular strip located on the dorsal face of the trachea was further dissected under binocular control. The epithelium was mechanically removed, and the epithelium-free muscular strip was cut into several pieces (1 × 1 mm) and incubated for 10 min in low-Ca2+ (200 µM) physiological saline solution (PSS, composition given below). The tissue was then incubated overnight (14 h) in low-Ca2+ PSS containing (in mg/ml) 0.5 collagenase, 0.35 pronase, 0.03 elastase, and bovine serum albumin at 4°C. After this time, the solution was removed, and the muscle pieces were incubated again in a fresh enzyme-free solution and triturated with a fire-polished Pasteur pipette to release cells. Cells were stored for 1-3 h to attach on glass coverslips at 4°C in PSS containing 0.8 mM Ca2+ and used on the same day.

Isometric Contraction Measurement

Isometric contraction was measured in tracheal rings that were mounted between two stainless steel clips in vertical 20-ml organ baths of a computerized isolated organ bath system (IOX; EMKA Technologies, Paris, France) previously described (29). Baths were filled with Krebs-Henseleit (KH) solution (composition in mM: 118.4 NaCl, 4.7 KCl, 2.5 CaCl2 · 2H2O, 1.2 MgSO4 · 7H2O, 1.2 KH2PO4, 25.0 NaHCO3, and 11.1 D-glucose, pH 7.4) maintained at 37°C and bubbled with a 95% O2-5% CO2 gas mixture. The upper stainless clip was connected to an isometric force transducer (EMKA Technologies). Tissues were set at optimal length by equilibration against a passive load of 1.5 g. Such a value has been previously determined for this type of preparation from normoxic rats (2). In preparations from hypoxic rats, the optimal passive load was determined by a series of length-tension experiments similar to that performed in normoxic animals, using ACh as contractile agonist. Briefly, after adjustment of the resting tension, each ring was stimulated by 10-3 M ACh (final concentration), and the maximal contractile force was recorded. The ring was then washed for its tension to return to the resting value, and the experiment was repeated for another resting tension. The tested resting tensions were 0.5, 1, 1.5, and 2 g, and the experiments were repeated on seven rings. The results indicate that a resting tension of 1.5 g is also the optimum passive load for CH rat tracheal rings.

At the beginning of each experiment, a supramaximal stimulation with ACh (10-3 M final concentration in the bath) was administered to each of the rings to elicit a reference response that was used to assess the viability of the tissue. Rings were then washed with fresh KH solution to eliminate the ACh response. When needed, tissues were then exposed to pollutants (see In Vitro Exposure to Pollutants). After washing the rings with KH solution, we constructed a cumulative concentration-response curve (CCRC) to carbachol. For comparison of the mechanical responsiveness of normoxic vs. CH rat tracheal rings, contractile response of each ring to each concentration of agonist was normalized to the smooth muscle cross-sectional area (SMA, mg/mm2) determined histologically (see Morphometric Studies) as well as expressed as a percentage of the maximal response. For assessment of the effect of pollutant exposure, the contractile response of each ring was expressed as a percentage of the maximal reference response in that ring.

Fluorescence Measurement and Estimation of [Ca2+]i

Changes in [Ca2+]i were monitored fluorimetrically using the Ca2+-sensitive probe indo 1 as previously described (28). Briefly, freshly isolated cells were loaded with indo 1 by incubation in PSS containing 1 µM indo 1-penta-acetoxymethyl ester (indo 1-AM) for 25 min at room temperature and then washed in PSS for 25 min. Coverslips were then mounted in a perfusion chamber and continuously superfused at room temperature. The recording system included a Nikon Diaphot inverted microscope fitted with epifluorescence (Nikon France, Charenton-le-Pont, France). Each cell was illuminated at 360 ± 10 nm, and the emitted light was counted simultaneously at 405 and 480 nm by two photomultipliers (P100, Nikon). [Ca2+]i was estimated from the 405/480 ratio (12) using a calibration for indo 1 determined within cells. PSS contained (in mM) 130 NaCl, 5.6 KCl, 1 MgCl2, 2 CaCl2, 11 glucose, and 10 HEPES, pH 7.4, with NaOH. ACh was applied to the tested cell by a 30-s pressure ejection from a glass pipette located close to the cell. No changes in [Ca2+]i were observed during test ejections of PSS (data not shown). Each record of [Ca2+]i response was obtained from a different cell. Each type of experiment was repeated on 21-68 cells from four different rats. [Ca2+]i responses of cells exposed to acrolein were expressed as a percentage of the mean response of unexposed cells from the same rat.

In Vitro Exposure to Pollutants

Exposure to pollutants was performed in tissues mounted in the organ bath system as previously described (29). After the ACh reference contraction and ACh washout, two of the four rings of each trachea were exposed to the pollutant for the desired period. The two unexposed rings served as paired temporal control. After pollutant administration, tissues were washed twice with KH solution, and 10 min after completion of pollutant exposure, the CCRC to carbachol was constructed. Duplicated rings were studied in each trachea, and a mean CCRC was calculated to be representative of that specimen.

Exposure to acrolein was performed for 5-40 min using acrolein in solution in the organ bath at a concentration of 0.3 µM (final concentration in the bath). For exposure to O3, a Teflon tube was attached to the vertical part of the L-shaped holder of the lower clip. The distal end of the Teflon tube formed a right angle so that it opened into the lumen of the airway rings when attached between the two clips. The proximal end of the Teflon tube was connected to an O3 generator (ozonator) coupled to an analyzer (photometer; ultraviolet photometric O3 calibrator 49PS; Thermoenvironmental Instruments, Franklin, MA). We have previously shown that exposure to clean air does not alter the subsequent response to agonists compared with that of unexposed tissues remaining immersed in KH solution (4).

Exposure of isolated cells to acrolein was performed as previously described (28). During the washing period after cell loading with indo 1, the coverslips with attached cells were immersed in PSS containing 0.2 and 0.3 µM acrolein for 10 min, while control coverslips remained in normal PSS. For the last 10 min of the washing period, exposed coverslips were immersed again in acrolein-free control PSS before examination.

Morphometric Studies

Subsequent to organ bath studies, rings were washed in KH solution, fixed, and embedded in Epon resin according to Hayat (13). Rings were fixed overnight in a solution containing 2% glutaraldehyde, 4.5% saccharose, 1 mM CaCl2, and 0.75 M Na-cacodylate pH 7.5. After washout and overnight postfixation in 1% osmium tetroxide, tissues were transferred into 1% uranyl aqueous solution, then dehydrated, and embedded in Epon.

Transversal semithin sections ~1- to 2-µm thick, perpendicular to the long axis of the trachea, were stained with Unna blue. Digitized images of the sections (×40 and ×100 magnifications) were obtained with an Optiphot microscope (Nikon, Tokyo, Japan) connected to a video camera module and to a microcomputer equipped with the Quancoul software (Quant'Image). SMA (mm2) and epithelium-plus-submucosa thickness (ESMT, µm) were determined on digitized images (×40 and ×100 magnifications, respectively) using Scion Image analysis software (Scion, Frederick, MD). For each ring, SMA was the mean value of data from four sections collected at different levels separated by ~15-20 µm, and ESMT was the mean value of eight measurements from two nonconsecutive sections.

Data Analysis and Statistics

Data are given as means ± SE. CCRC were fitted by a nonlinear regression equation
F<IT>/</IT>F<SUB>max</SUB><IT>=</IT>1<IT>/</IT>[1<IT>+e</IT><SUP><IT>−</IT>2,3n(logC<IT>+</IT>pEC<SUB>50</SUB>)</SUP>]
where F is the force, Fmax the maximal force, C the concentration, EC50 the concentration producing half-maximal force (pEC50 -logEC50), and n the Hill coefficient. Change in airway reactivity induced by pollutant exposure was defined as Delta Fmax, the difference between Fmax in exposed an unexposed tissues expressed as the percentage of Fmax in the control rings. Differences in Fmax and EC50 were tested using nonlinear regression analysis according to Meddings et al. (23). Statistical comparisons of [Ca2+]i response between cells unexposed and exposed to acrolein were carried out by Mann-Whitney U-tests for quantitative variables and chi 2-tests for qualitative variables. The effects of CH on rat weight, SMA, ESMT, and ACh response were assessed by Student's t-tests. Results were considered significant at P < 0.05.

Chemicals and Drugs

ACh, carbachol, pronase (type E), elastase (type 3), bovine serum albumin, glutaraldehyde, and acrolein (minimum 90%, stabilized with 0.1-0.2% hydroquinone) were purchased from Sigma (St-Quentin Fallavier, France). Collagenase (type CLS1) was from Worthington Biochemical (Freehold, NJ). Osmium tetroxide, oxide propylene, and Epon were obtained from Merck Eurolab (Pessac, France), and uranyl acetate was from Euromedex (Souffelwegershein, France). Indo 1-AM was from Calbiochem (France Biochem, Meudon, France).

Indo 1-AM was dissolved in dimethyl sulfoxide (DMSO). The maximal concentration of DMSO used in our experiments was <0.1% and had no effect on the resting value of the [Ca2+]i nor on the variation of the [Ca2+]i induced by ACh (data not shown).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Pollutant Exposure on Trachea Responsiveness in CH Rats

Exposure to acrolein. In this series of experiments, we assessed the effect of in vitro preexposure to acrolein (0.3 µM) for 5, 10, 20, and 40 min on the contractile response of hypoxic rat tracheal rings to carbachol. Each experimental procedure was repeated on six different hypoxic rat tracheae. Preexposure to acrolein for any duration did not significantly modify the responsiveness of CH tracheae to carbachol. Table 1 indicates the values of Fmax and pEC50 in unexposed and exposed tissues. In Fig. 1A, change in airway reactivity, expressed as Delta Fmax, is plotted against the product of exposure concentration and exposure time, i.e., a surrogate of the dose of acrolein. Whatever the dose, Delta Fmax did not significantly differ from zero.

                              
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Table 1.   Fmax and pEC50 in hypoxic rat tracheal rings exposed and unexposed to acrolein and ozone



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Fig. 1.   Effect of pollutant exposure on isometric contraction to carbachol in chronically hypoxic (CH) rat trachea. A: effect of exposure to acrolein, expressed as the product of concentration (C) and exposure time (T), in µM · min. B: effect of exposure to ozone in parts/million · min. Delta Fmax, change in maximal force induced by acrolein exposure. Each symbol is mean value from 6 specimens. Vertical bars are SE.

Exposure to ozone. The effect of in vitro preexposure to ozone (1 part/million) for 10, 15, and 20 min was also examined on the contractile response of hypoxic rat tracheal rings to carbachol. Each experimental procedure was repeated on six different rat tracheae. As for acrolein, preexposure to ozone for any duration did not significantly modify the responsiveness of CH trachea to carbachol. Results are given in the table and shown in Fig. 1B.

Acrolein exposure of epithelium-free tracheal rings. To verify that the absence of effect of the pollutants on airway responsiveness from CH rats was not related to a limited accessibility of the pollutant to airway smooth muscle, we assessed the effect of acrolein in epithelium-free hypoxic tracheal rings. Exposure to acrolein was performed at 0.3 µM for 10 min, a dose that induces the maximal hyperresponsiveness in normoxic rat tracheal rings (28). In contrast with these previous results, the CCRC was not significantly altered by preexposure to acrolein in tracheae from CH rats.

Effect of Acrolein Exposure on [Ca2+]i Response to ACh in Isolated Myocytes from CH Rats

In these experiments, we assessed the effect of acrolein on both the resting calcium concentration and the calcium response to low (0.1 µM) and high (10 µM) concentrations of ACh in tracheal myocytes freshly isolated from CH rats. Original traces are shown in Fig. 2. Cells were exposed to two different acrolein concentrations, i.e., 0.2 and 0.3 µM, for 10 min. These conditions of exposure to acrolein have been shown to increase the calcium response to both low and high ACh concentrations in freshly isolated myocytes from normoxic rats (28).


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Fig. 2.   Typical traces of intracellular Ca2+ concentration ([Ca2+]i) response to ACh in myocytes freshly isolated from CH rat tracheae unexposed and exposed to acrolein. A: effect of a 30-s ejection of 0.1 µM ACh on [Ca2+]i in myocytes unexposed (control) and exposed to 0.2 or 0.3 µM acrolein for 10 min. B: effect of a 30-s ejection of 10 µM ACh on [Ca2+]i in myocytes unexposed (control) and exposed to 0.2 or 0.3 µM acrolein for 10 min. Each trace is representative from 21-68 cells.

In unexposed cells, the mean resting [Ca2+]i was 123 ± 2.7 nM (n = 110). Preexposure to 0.2 and 0.3 µM acrolein for 10 min did not modify the resting [Ca2+]i values, which were -0.62 ± 2.68% (n = 73) and -5.85 ± 2.92% of control (n = 66), respectively.

Stimulation of the unexposed cells by a 30-s ejection of 0.1 µM ACh (n = 46) caused in 73% of the cells a transient Ca2+ rise, the mean value of which was 298 ± 3.7 nM. In 27% of the responding cells, this first peak was followed by [Ca2+]i oscillations whose mean frequency was 6.0 ± 1.0 oscillations/min. Preexposure to acrolein, whatever the concentration, did not significantly modify the percentage of responding cells or the percentage of oscillating cells. In contrast, the mean [Ca2+]i rise induced by 0.1 µM ACh was significantly increased by 65.8 ± 27.08 and 39.7 ± 12.89% compared with control after a 10-min exposure of the cells to 0.2 (n = 21) and 0.3 (n = 25) µM acrolein, respectively (Fig. 3A). The mean oscillation frequency was also enhanced by 34.7 ± 61.45 and 52.4 ± 21.38%, respectively, but in a nonsignificant manner. The lack of statistical significance is due to the fact that, at low ACh concentration, in the present study as in our previous ones (27, 28), calcium responses exhibit larger interindividual variations on stimulation than at high ACh concentration, which corresponds to greater variance and SE.


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Fig. 3.   Effect of acrolein exposure on [Ca2+]i response to ACh in myocytes freshly isolated from CH rat tracheae. A: effect of 10-min exposure to acrolein on [Ca2+]i response to low (0.1 µM) ACh concentration. Left: percentage of responding cells unexposed (solid bar) and exposed (open bars) to 0.2 and 0.3 µM acrolein. Right: effect of acrolein on ACh-induced [Ca2+]i rise (maximum increase above baseline) expressed as the percentage of ACh-induced [Ca2+]i rise in control cells. B: effect of 10-min exposure to acrolein on [Ca2+]i response to high (10 µM) ACh concentration. Left: effect of acrolein on ACh-induced [Ca2+]i rise (maximum increase above baseline) expressed as the percentage of ACh-induced [Ca2+]i rise in control cells. Right: effect of 10-min acrolein exposure on oscillation frequency, expressed as the percentage of the oscillation frequency in control cells. Vertical bars are SE. * P < 0.05.

Stimulation of unexposed cells by 10 µM ACh (n = 68) induced, in 97% of the cells, a first [Ca2+]i peak of 471 ± 30.3 nM followed, in 50% of the responding cells, by [Ca2+]i oscillations with a mean frequency of 10.5 ± 0.8/min. Preexposure to 0.2 (n = 56) and 0.3 (n = 41) µM acrolein did not significantly modify the percentage of responding cells, the percentage of oscillating cells, or the value of the first [Ca2+]i rise. In contrast, it significantly increased the oscillation frequency by 25.0 ± 5.6 and 37.3 ± 9.6%, respectively (Fig. 3B).

Effect of CH on Trachea Structure and Reactivity

To identify a possible effect of CH on airway smooth muscle that may explain the differential effect of pollutant exposure in tissues from CH rats vs. normoxic ones, we further investigated the effect of CH alone on the responsiveness of rat tracheal rings to muscarinic agonists. The sensitivity was quantified by the pEC50 calculated from CCRC to carbachol normalized to the maximal contractile response and fitted by nonlinear equation (Fig. 4A). Because CH may alter the proportion of smooth muscle within the tissue, to obtain a precise determination of smooth muscle maximal contractility in CH vs. normoxic tracheae, we normalized the Fmax in response to cholinergic agonists to SMA. Histological studies, morphometric analysis, and smooth muscle contractile ACh response were performed on 23 rings from six different normoxic rat tracheae and 27 rings from seven different CH rat tracheae. CCRC to carbachol were performed on 11 normoxic rings from six different rats and 13 hypoxic rings from seven rats.


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Fig. 4.   Effect of CH on airway responsiveness. A: effect of CH on the cumulative concentration-response curve to carbachol. X-axis: log concentration of carbachol. Y-axis: contractile response to carbachol normalized to maximal contraction in normoxic (, n = 6 rats) and in CH (, n = 7 rats) rat trachea. Experimental data are fitted by nonlinear equation described in MATERIALS AND METHODS. B: effect of CH on maximal contractile response to 10-3 M ACh, normalized to smooth muscle cross-sectional area (SMA, mg/mm2) in normoxic (sold bar, n = 23) and in CH (open bar, n = 27) rat tracheal rings. * P < 0.05. Vertical bars are SE.

In accordance with our previous studies (1), CH increased the sensitivity of tracheal rings to muscarinic stimulation. Calculated pEC50 values were 6.40 ± 0.06 and 6.24 ± 0.05 M in rings from hypoxic and normoxic animals, respectively (P < 0.05). In addition, the slope of the curve was significantly decreased in hypoxic animals, the Hill coefficient being 1.00 ± 0.26 and 1.22 ± 0.06 in hypoxic and normoxic tissues, respectively (P < 0.05). In contrast, maximal contractile response to a cholinergic agonist was significantly decreased in hypoxic rats. Maximal responses of tracheal rings to supramaximal concentration of ACh (10-3 M) normalized to SMA were 43,818 ± 4,325 mg/mm2 in normoxic rings and 31,733 ± 2,776 mg/mm2 in hypoxic ones (Fig. 4B). Similarly, nonlinear regression analysis of the CCRCs showed that the maximal response to cumulative concentrations of carbachol (Fmax) was significantly lowered in hypoxic vs. normoxic trachea (41,339 ± 4,625 and 67,093 ± 8,851 mg/mm2, respectively, P > 0.05). Finally, CH did not significantly modify trachea histology. SMA was smaller in hypoxic tissues (104,077 ± 5,819 µm2) than in normoxic ones (122,239 ± 13,075 µm2), but the difference was not significant. In addition, ESMT was similar in hypoxic (77.7 ± 2.9 µm) and in normoxic (77.1 ± 2.8 µm) trachea rings.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study indicates that, in contrast with previous results obtained in airway tissue from normoxic rats, in vitro exposure to pollutants such as acrolein or ozone does not alter cholinergic agonist-induced contraction in CH rats. This lack of effect on airway mechanical activity cannot be ascribed to a lack of effect on calcium signaling at the site of the smooth muscle cell, since acrolein-induced increase in ACh-evoked [Ca2+]i response in airway myocytes from CH rats appeared similar to that in cells from normoxic rats. We therefore suggest that the complex effect of CH on calcium-contraction coupling (i.e., hypersensitivity and hyporeactivity) accounts for the apparent inability to produce airway hyperresponsiveness under such experimental conditions.

In contrast with previous results that demonstrated that atmospheric pollutants such as NO2, ozone, or acrolein alter airway reactivity, this study revealed that ozone and acrolein have no apparent effect on airway mechanical responsiveness. In normoxic airways, we have shown that changes in reactivity depends on the dose of pollutant, i.e., the product of the concentration and the duration of exposure. In rat trachea as well as in human bronchi, pollutant-induced change in reactivity (Delta Fmax), when plotted against the dose of pollutant, exhibits a bell-shaped curve (2, 3, 26). It is unlikely that the lack of effect of ozone and acrolein on airways from CH rats is due to a shift in the dose-effect curve toward low or high doses, since these pollutants have been tested over a wide range of doses. Also, this absence of effect does not seem to involve changes in smooth muscle accessibility to the pollutants. Contractile response of epithelium-free tracheal rings from CH rats was also not modified by exposure to a dose of acrolein that induces hyperreactivity in epithelium-free trachea from normoxic animals (28). Moreover, histological studies have confirmed that ESMT, whose changes may alter smooth muscle accessibility to pollutants, was not modified by CH.

Although exposure to acrolein had no effect on airway contraction, it did enhance the cholinergic-induced calcium response in freshly isolated myocytes from CH rats in a way similar to that observed in airway smooth muscle cells from normoxic animals. It has been previously shown in airway smooth muscle cells from various species that the amplitude of the first peak and/or the oscillation frequency depends on ACh concentration (18, 19, 24, 25). The effect of increasing agonist concentrations appears to be different at the subcellular vs. the whole cell level. At the subcellular level, the amplitude of regional [Ca2+]i oscillations remains constant, whereas the frequency increases with ACh concentration (25). At the whole cell level, we have previously shown that the global [Ca2+]i oscillation frequency and [Ca2+]i rise in amplitude depend on ACh concentration and that the increase in both the amplitude of the first peak and the amplitude of the frequency of oscillations contributes to the graded increase in the mechanical response over the wide range of cholinergic stimulation, i.e., from 0.1 up to 10 µM ACh concentrations (27). We previously demonstrated in control myocytes that acrolein had no effect on the [Ca2+]i resting value but increased the Ca2+ response to low and high ACh concentrations. More precisely, at low (0.1 µM) ACh concentration, acrolein increased the amplitude of the first Ca2+ peak, without significantly altering the percentage of responding cells or that of oscillating ones. At high ACh concentrations, acrolein did not modify the amplitude of the first [Ca2+]i rise but increased the frequency of oscillations. This acrolein-dependent effect on the calcium response accounts for acrolein-induced hyperreactivity observed in contractile experiments conducted in normoxic animals (27).

The apparent lack of effect of pollutants to produce in vitro mechanical hyperresponsiveness in airway tissue from CH rats, despite a marked effect on calcium signaling similar to that observed in normoxic animals, is likely to be related to the effect of CH per se on calcium-contraction coupling in airway smooth muscle. Previous studies that have evaluated the effect of CH on rat tracheal hyperresponsiveness produced conflicting results. Clayton et al. (6, 7) reported that CH attenuates contractile responses in rat airways in vitro, whereas Belouchi et al. (1) indicated that CH increases calcium responses in rat tracheal myocytes. In the present study, we have reexamined the effect of CH on the mechanical activity of rat isolated tracheal muscle with special attention to the normalization of the isometric force, since CH may induce smooth muscle remodeling (10, 14). In agreement with our previous study, we have observed that CH increases the sensitivity to cholinergic agonists, a finding that can be ascribed to the effect of CH on calcium signaling (1). By contrast, CH markedly decreased the maximal contractile response normalized to SMA. This finding is in agreement with that of Clayton et al. (6) obtained in similar hypoxic conditions in young rats (28- to 30-days old). The fact that CH enhances calcium signaling and decreases airway reactivity strongly indicates that it decreases the calcium sensitivity of the contractile apparatus. This phenomenon is likely to explain the overall effect of ozone in the present study.

In conclusion, our study shows that exposure to air pollutants does not induce airway hyperresponsiveness in CH rats, in contrast with normoxic rats, although it increases the calcium response to both low and high concentrations of ACh in isolated myocytes. These discrepancies cannot be explained by change in smooth muscle accessibility but may be linked to alteration of calcium-contraction coupling caused by CH, which has complex effects on airway smooth muscle contractility, inducing hyporeactivity and hypersensitivity. The clinical implication of the present findings, i.e., the effect of air pollutant on bronchial responsiveness in CH patients (COPD), should now be assessed.


    ACKNOWLEDGEMENTS

The authors are grateful to Hugette Crevel, Christiane Salat, and Pierre Téchoueyres for technical assistance.


    FOOTNOTES

This study was supported by grants from the Ministère de l'environnement and Agence de l'environnement et de la maîtrise de l'énergie (ADEME PRIMEQUAL no. 99 62 035).

Address for reprint requests and other correspondence: E. Roux, Laboratoire de Physiologie Cellulaire Respiratoire, INSERM EMI 9937, Université Victor Segalen Bordeaux 2, 146 rue Léo-Saignat, 33076 Bordeaux cedex, France (E-mail: etienne.roux{at}u-bordeaux2.fr).

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.

April 19, 2002;10.1152/ajplung.00387.2001

Received 2 October 2001; accepted in final form 11 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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