Introduction

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Acrolein, a potent respiratory irritant, is an unsaturated aliphatic aldehyde emitted in the environment by automobile exhaust, cigarette smoke, and the burning of wood and fat-containing foods (1). The use of oxygenated fuel to improve air quality is being discussed although it may, as a side effect, increase the outdoor exposure to aldehydes (2). When inhaled, acrolein as well as other pollutants induce airway hyperresponsiveness in a variety of species (3). For example, in guinea pigs, inhalation of acrolein at a concentration in the range of 1 ppm increases pulmonary resistance and bronchial responsiveness to acetylcholine (7). This enhancement in bronchial reactivity caused by air pollutants present in the environment poses a serious human health concern, especially in patients suffering from obstructive airway diseases or sensitized by allergies.

We have previously reported that a variety of pollutants, administered ex vivo to the lung, alters the subsequent in vitro mechanical responsiveness of airway smooth muscle (8). In particular, we have observed that acrolein exposure increases the reactivity of human bronchial and rat tracheal rings to carbachol in a dose-dependent manner (9, 10). Similar results have been obtained with O₃ (11). Our previous studies, as well as those conducted in a variety of species (6, 12), have provided indirect evidence that these pollutants may share, among other mechanisms (15), a common action at the site of calcium homeostasis in airway smooth muscle. Relevant to this is the observation that a direct effect of O₃ on cytosolic Ca²⁺ concentration ([Ca²⁺]_i) homeostasis has been demonstrated in human tracheal epithelial cells (16). The release of intracellular calcium can now be directly studied by microspectrofluorimetry using fluorescent dyes in isolated airway myocytes. We recently characterized the variations in [Ca²⁺]_i in response to muscarinic stimulation of smooth muscle cells freshly isolated from the rat trachea. These variations corresponded to a special pattern, the so-called Ca²⁺ oscillations, the amplitude and frequency of which depend on the cholinergic agonist concentration (17).

The aim of the present study was thus to determine the effect of in vitro exposure to acrolein of rat isolated airway tracheal myocytes on cholinergic-induced [Ca²⁺]_i response. We also compared the range of doses of acrolein altering [Ca²⁺]_i responses in isolated airway smooth muscle cells with that required to increase cholinergic-induced isometric contraction of rat epithelial-denuded tracheal rings.

	Materials and Methods

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Tissue Preparation

Rat tracheae were obtained from male Wistar rats 10 to 15 wk old, weighing 300 to 400 g. For each experiment, a rat was anesthetized by intraperitoneal administration of 400 mg ethylcarbamate. Heart and lungs were removed en bloc, and the trachea was rapidly dissected out. For isometric contraction measurements, the trachea was cut into four rings of similar 3-mm diameter and 3 to 4 mm in length, as previously described (9). The epithelium was then mechanically removed 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 histologic examination of 2-µm thin sections of tissues embedded in glycolmethacrylate resin at the end of the contraction experiments (not shown). For fluorescence measurements of [Ca²⁺]_i in isolated cells, the muscular strip located on the dorsal face of the trachea was further dissected under binocular control, as previously described (17). 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) physiologic saline solution (PSS; composition given below). The tissue was then incubated in low-Ca²⁺ PSS containing 1.0 mg · ml¹ collagenase, 0.7 mg · ml¹ pronase, 0.06 mg · ml¹ elastase, and 3 mg · ml¹ bovine serum albumin (BSA) at 37°C for two successive periods of 25 min. 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 to 3 h to attach on glass coverslips at 4°C in PSS containing 0.8 mM Ca²⁺ and used on the same day.

Fluorescence Measurement and Estimation of [Ca²⁺]_i

Changes in [Ca²⁺]_i were monitored fluorimetrically using the Ca²⁺-sensitive probe indo-1 as previously described (17). 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. Exposure of isolated cells to acrolein was performed during this washing period by immersing the coverslips with attached cells in PSS containing a variety of acrolein concentrations ranging from 0.1 to 1 µM for durations of exposure from 5 to 15 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. 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). A single cell was illuminated at 360 ± 10 nm. Emitted light from a window, which was slightly larger than the cell and was manually adjusted to the size of each of the tested cells, was counted simultaneously at 405 nm and 480 nm by two photomultipliers (P100; Nikon). Voltage signals at each wavelength were stored in an IBM-PC computer for subsequent analysis. The fluorescence ratio (405/480) was calculated on-line and displayed with the two voltage signals on a monitor. [Ca²⁺]_i was estimated from the 405/480 ratio (18) using a calibration for indo-1 determined within cells (19).

The PSS contained (in mM): 130 NaCl, 5.6 KCl, 1 MgCl₂, 2 CaCl₂, 11 glucose, and 10 N-2-hydroxyethylpiperazine- N'-ethane sulfonic acid, pH 7.4, with NaOH. Acetylcholine (ACh) or caffeine 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). Generally, each record of [Ca²⁺]_i response to ACh or caffeine was obtained from a different cell. Each type of experiment was repeated for the number of cells indicated in the text.

Isometric Contraction Measurement

Isometric contraction was measured in airway smooth muscle rings that were mounted between two stainless-steel clips in vertical 20-ml organ baths of a computerized isolated organ bath system (IOS₁; EMKA Technologies, Paris, France) previously described (8). Baths were filled with Krebs-Henseleit solution (composition in mM: 118.4 NaCl, 4.7 KCl, 2.5 CaCl₂ · 2 H₂O, 1.2 MgSO₄ · 7 H₂O, 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, as previously determined for this type of preparation (9). At the beginning of each experiment, a supramaximal stimulation with ACh (10³ M final concentration in the bath) was administered to each of the rings to elicit a reference response. After washing the rings with fresh Krebs-Henseleit solution to eliminate the ACh response, two of the rings were exposed to a solution containing 0.3 µM acrolein during times varying between 5 and 45 min. The unexposed rings served as temporal controls. At completion of exposure, all four rings were washed twice to remove unabsorbed acrolein and a cumulative concentration-response curve (CCRC) to carbachol (10⁸ to 10⁴ M) was constructed.

Chemicals and Drugs

Collagenase (type CLS1) was from Worthington Biochemical Corp. (Freehold, NJ). Acrolein, minimum 90% pure and stabilized with 0.1% hydroquinone, pronase (type E), elastase (type 3), BSA, ACh, and caffeine were purchased from Sigma (Saint Quentin Fallavier, 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).

Data Analysis and Statistics

Results of [Ca²⁺]_i are expressed as the mean ± standard error of the mean (SEM) with n the number of cells of the sample. In each rat, the mean values of both control cells and cells exposed to acrolein were calculated to be representative of that rat. Each experiment was replicated on four different rats, and statistical comparisons were carried out using Student's paired t tests.

In contraction experiments, the contractile response to each ring was expressed as a percent of the maximal reference ACh response in that ring. Since duplicate airway rings were studied in each experimental condition from the individual CCRC constructed in each ring, a mean CCRC was obtained for the two rings (either control or test) to be representative of that trachea and repeated on five to six different specimens. Overall mean CCRC were generated in control and test tissues and compared. The plateau of the contractile force on the CCRC (i.e., F_max) is expressed as the mean ± SEM. The EC₅₀ (the concentration of agonist producing 50% of the maximal response) was calculated using a least-squares linear regression method and overall results are expressed as geometric means with 95% confidence limits. The change in airway smooth muscle responsiveness was defined as F_max, i.e., the difference between F_max in test and control rings expressed as a percentage of F_max in the control ring. Statistical comparison of paired mean CCRC was carried out using first an analysis of variance (two-way ANOVA) for two or three variables along the whole curve to determine whether the curves were different from each other. Then, when the F test was significant, modified Student's paired t tests (two-tailed) using the Bonferroni correction were carried out to find out the concentrations for which the responses were statistically different (20). Results were considered significant at P < 0.05.

	Results

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Effect of Acrolein Exposure on [Ca²⁺]_i Response to ACh in Isolated Tracheal Myocytes

In this series of experiments, we assessed the effect of acrolein on both the resting calcium concentration and the calcium response to low (0.1 µM) or high (10 µM) concentrations of ACh in freshly isolated smooth muscle cells. Cells were exposed to a variety of acrolein concentrations from 0.1 to 1 µM and durations from 5 to 15 min.

In unexposed cells, the mean resting concentration of Ca²⁺ was 128 ± 2.9 nM (n = 133). Pre-exposure to the fixed concentration of 0.2 µM acrolein for 5, 10, or 15 min did not modify the resting [Ca²⁺]_i values which were 136 ± 3.5 (n = 103), 127 ± 3.4 (n = 123), and 126 ± 3.3 nM (n = 108), respectively. Moreover, direct ejection for 30 s of 0.2 µM acrolein failed to induce any change in [Ca²⁺]_i (n = 10, data not shown).

Stimulation by a 30-s ejection of 0.1 µM ACh (low concentration) caused, in 71% of the unexposed myocytes tested (n = 71), a transient increase in [Ca²⁺]_i for which the mean value was 195 ± 26 nM (Figure 1a). In 21% of the responding cells, the first [Ca²⁺]_i rise was followed by [Ca²⁺]_i oscillations. Exposure to a fixed concentration of 0.2 µM acrolein for 5 (n = 35), 10 (n = 45), and 15 min (n = 37) did not significantly modify the percentage of responding cells. Similarly, the percentage of oscillating responses (9.1, 28.6, and 24%, respectively) was not altered by the pollutant. In contrast, the mean [Ca²⁺]_i rise induced by 0.1 µM ACh was significantly enhanced by 50.8 and 77% compared with control after exposure for 5 and 10 min, respectively (Figure 2a). Likewise, when the duration of exposure was kept constant (10 min), increasing the concentration of acrolein did not significantly modify the percentage of responding cells but did increase the mean [Ca²⁺]_i rise induced by 0.1 µM ACh by 49.4% for 0.3 µM (n = 48) (Figure 2b).

View larger version (24K): [in this window] [in a new window]   Figure 1.   Effect of acrolein exposure on [Ca2+]i response to ACh in myocytes freshly isolated from rat trachea. (a) Effect of a 30-s ejection of 0.1 µM ACh on [Ca2+]i in smooth muscle cells unexposed (control) or pre-exposed to a fixed concentration of acrolein (0.2 µM) for various times indicated on the records. (b) Effect of a 30-s ejection of 10 µM ACh on [Ca2+]i in smooth muscle cells unexposed (control) or pre-exposed to a fixed concentration of acrolein (0.2 µM) for various times indicated on the records. Each trace is representative of 35 to 78 different cells.

View larger version (23K): [in this window] [in a new window]   Figure 2.   Effect of acrolein exposure on [Ca2+]i response to 0.1 µM ACh in myocytes freshly isolated from rat trachea. (a) Effect of varying the duration of exposure to a fixed concentration of acrolein (0.2 µM); left panel: percentage of responding cells unexposed (solid column) or pre-exposed (open columns) to acrolein. Right panel: Effect of acrolein on ACh-induced [Ca2+]i rise (maximal increase above baseline concentration), expressed as the percentage of ACh-induced [Ca2+]i rise in control cells. (b) Effect of varying the concentration of acrolein for a fixed duration of exposure (10 min); left panel: percentage of responding cells unexposed (solid column) or pre-exposed (open columns) to acrolein. Right panel: Effect of acrolein on ACh-induced [Ca2+]i rise (maximal increase above baseline concentration), expressed as the percentage of ACh-induced [Ca2+]i rise in control cells. Vertical bars are SEM. *P < 0.05.

Stimulation of unexposed myocytes with a high concentration of ACh (10 µM) induced in 100% of the cells a first [Ca²⁺]_i rise of a mean value of 577 ± 35 nM (n = 62) followed, in 53.2% of the cases, by oscillations with a mean frequency of 9.3 ± 0.7/min (Figure 1b). Exposure to a fixed concentration of acrolein (0.2 µM) for 5 (n = 68), 10 (n = 78), and 15 min (n = 71) did not significantly alter the first [Ca²⁺]_i peak or the percentage of responding cells, which were 58.8, 54.9, and 56.3%, respectively. In contrast, acrolein exposure increased the frequency of oscillations by 44.4% for 10 min (Figure 3a). Similarly, when the duration of exposure was kept constant (10 min), increasing the concentration of acrolein also increased the frequency of oscillations induced by 10 µM ACh by 36.3% for 0.3 µM (n = 16) (Figure 3b).

View larger version (24K): [in this window] [in a new window]   Figure 3.   Effect of acrolein exposure on [Ca2+]i response to 10 µM ACh in myocytes freshly isolated from rat trachea. (a) Effect of varying the duration of exposure to a fixed concentration of acrolein (0.2 µM); left panel: ACh-induced first [Ca2+]i rise (maximal increase above baseline concentration) (in nM) in cells unexposed (solid column) or pre-exposed (open columns) to acrolein; right panel: effect of acrolein on oscillation frequency, expressed as the percentage of the oscillation frequency in control cells. (b) Effect of varying the concentration of acrolein for a fixed duration of exposure (10 min); left panel: ACh-induced first [Ca2+]i rise (maximal increase above baseline concentration) (in nM) in cells unexposed (solid column) or pre-exposed (open columns) to acrolein; right panel: effect of acrolein on oscillation frequency, expressed as the percentage of the oscillation frequency in control cells. Vertical bars are SEM. *P < 0.05.

Effect of Acrolein Exposure on [Ca²⁺]_i Response to Caffeine in Isolated Tracheal Myocytes

In this series of experiments, we investigated the consequence of acrolein exposure (0.1-1 µM) on the response to caffeine, an agent that also increases intracellular Ca²⁺ concentration in airway smooth muscle. We first constructed a concentration-response curve to caffeine from 10 µM to 5 mM (Figure 4c). Whatever the duration of caffeine ejection, stimulation of rat tracheal smooth muscle cells induced a single transient rise, the amplitude of which gradually increased with the concentration. The maximal increase in [Ca²⁺]_i was 698 ± 59 nM (n = 19) for 5 mM caffeine and the EC₅₀ was 0.25 mM. A 10-min exposure to acrolein did not alter the caffeine (0.1 mM)-induced [Ca²⁺]_i response whatever the acrolein concentration (Figures 4a and 4b).

View larger version (21K): [in this window] [in a new window]   Figure 4.   Effect of acrolein exposure on [Ca2+]i response to caffeine in myocytes freshly isolated from rat trachea. (a) Effect of a 30-s ejection of 0.1 mM caffeine on [Ca2+]i in smooth muscle cells unexposed (control) or pre-exposed to 0.2 µM acrolein for 10 min. Each trace is representative of 47 to 57 different cells. (b) Caffeine-induced [Ca2+]i rise according to acrolein concentration expressed as a percentage of caffeine-induced [Ca2+]i in control (unexposed) cells. Vertical bars are SEM. (c) Non-cumulative concentration-dependent effect of caffeine on [Ca2+]i response in rat tracheal myocytes. Abscissa: concentration of caffeine (log M). Ordinate: [Ca2+]i rise (maximal increase above baseline concentration) (in nM). Each point of the curve represents a mean value calculated from n = 11 to 24 different cells. Vertical bars are SEM.

Effect of Acrolein Exposure on Isometric Contraction in Epithelium-free Tracheal Rings

In these experiments, we assessed the effect of acrolein exposure at the fixed concentration of 0.3 µM for various durations on isometric contraction of epithelium-free tracheal rings induced by muscarinic stimulation. Exposure to acrolein did induce hyperresponsiveness to carbachol in rat tracheal rings, and the maximal effect was observed for a 10-min exposure time. For a 20-min exposure, the effect of acrolein on epithelium-free tracheal rings was less pronounced, though still significant. With longer exposure, the responsiveness of exposed rings decreased and became equal to, or even less than, that of unexposed rings (Table 1). When plotted against a surrogate of the "dose," i.e., the product of the concentration of acrolein (C) by the duration of exposure (T), the change in carbachol-induced responsiveness, defined as F_max, exhibits a bell-shaped curve, as shown in Figure 5, and obeys Haber's law (21).

View larger version (12K): [in this window] [in a new window]   Figure 5.   Effect of exposure to acrolein on carbachol-induced isometric contraction in rat epithelium-free tracheal rings. Abscissa: dose of acrolein, expressed as the product of acrolein exposure concentration (C) in µM and exposure time (T) in min. Ordinate: change in maximal contraction to carbachol relative to control (Fmax). Each symbol represents a mean value calculated from n = 5 to 7 different specimens. Vertical bars are SEM. *P < 0.05.

	Discussion

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The present study indicates that exposure to acrolein of freshly isolated airway myocytes alters cholinergic-induced [Ca²⁺]_i responses. Acrolein increased the amplitude of the first [Ca²⁺]_i peak in response to a low ACh concentration and the frequency of [Ca²⁺]_i oscillations induced by a high ACh concentration. The effect of altering the duration of exposure to a fixed concentration of acrolein on Ca²⁺ signaling was similar to that of altering the concentration of the pollutant for a fixed duration of exposure. The "dose" of acrolein (estimated as the product of the concentration of acrolein by the duration of exposure) that potentiated calcium signaling in isolated myocytes was in the same range as that increasing isometric contraction to cholinergic agonist in epithelial-denuded rings from the same rat tracheal preparation. Finally, the effect of acrolein on calcium homeostasis was not observed in response to caffeine, an alternative Ca²⁺ releaser agent in airway smooth muscle.

Previous studies have investigated the mechanisms of action of gas pollutants on airway hyperresponsiveness at the site of the airway smooth muscle (8). A first approach has been to examine the effect of pollutants on agonists producing airway contraction via different excitation-contraction coupling pathways. Acrolein, as O₃, did not alter the response to KCl, suggesting that such pollutants had no effect on the electromechanical coupling of airway smooth muscle, that is, on the contractile activity that depends on surface membrane potential changes. Conversely, these pollutants increased the contractile responses to agonists that, as part of their mechanism of action, produce contraction via activation of pharmacomechanical coupling (i.e., a coupling that is independent of changes in the membrane potential of the smooth muscle cell), such as cholinergic agonists. Although some of the effects of cholinergic agonists depend on membrane potential changes, these results have led to the hypothesis that pollutants may interact with the release of intracellular calcium ions. This hypothesis has been supported by experiments performed in calcium-free medium, showing that removal of external calcium does not prevent the O₃-induced increase in isolated airway responsiveness when O₃ is administered either in vivo to animals prior to in vitro experiments (13) or directly ex vivo (11).

Recent experiments have revealed, however, that [Ca²⁺]_i changes in response to muscarinic stimulation of freshly isolated airway smooth muscle cells constitute a complex signal, the so-called [Ca²⁺]_i oscillations (17, 22- 24). The amplitude of the first [Ca²⁺]_i rise is graded at low ACh concentrations (< 0.2 µM). Oscillations occur in response to higher ACh concentrations (> 0.2 µM) and their frequency increases with ACh concentration (up to 100 µM). It has thus been suggested that the amplitude of the physiologic response, that is, the mechanical activity induced by cholinergic stimulation, depends on the amplitude of the first [Ca²⁺]_i rise at low concentrations and then on the oscillation frequency at high cholinergic concentrations (17, 23, 25). For this reason, we examined the effect of acrolein on intracellular calcium release in response to both a low (0.1 µM) and a high (10 µM) concentration of ACh.

We observed that, whatever the ACh concentration, acrolein strongly affected calcium signaling in airway smooth muscle. In response to a low ACh concentration, acrolein increased the amplitude of the first [Ca²⁺]_i peak, suggesting that it interacts with any of the steps coupling muscarinic cholinoceptor activation to the opening of the IP₃ receptor-Ca²⁺ channel in the sarcoplasmic reticulum and/or the Ca²⁺ content of this internal store because these two latter phenomena determine the amplitude of the first [Ca²⁺]_i peak (17). Acrolein also altered the second component, i.e., Ca²⁺ oscillations, of the agonist-induced calcium response, which is better analyzed at high ACh concentrations. As a general rule, in cultured airway smooth muscle cells, the secondary steady-state phase of the Ca²⁺ response is a sustained phase that often depends on an influx of extracellular calcium (26). However, in freshly isolated airway smooth muscle cells, [Ca²⁺]_i oscillations have been described by several groups in response to cholinergic agonists (23- 25, 29, 30). Whatever the species, these oscillations have the following common characteristics: (1) they are primarily IP₃-dependent, (2) they involve a cyclic Ca²⁺ release-Ca²⁺ re-uptake by intracellular store, and (3) their frequency increases with the increase in the cholinergic agonist concentration. In the present study we have observed that, as for the first [Ca²⁺]_i peak, acrolein increased [Ca²⁺]_i oscillation frequency. On both components of the cholinergic-induced Ca²⁺ signal, the effect of altering the duration of exposure to a fixed concentration of acrolein was similar to that of altering the concentration of the pollutant for a fixed duration of exposure.

As discussed above, the effect of acrolein at both low and high ACh concentrations suggests that it interacts with receptor-mediated IP₃ signaling and/or Ca²⁺ releasing and re-uptake functions of the intracellular store. To examine further the effect of acrolein on these stores, we conducted experiments with caffeine. This agent also causes Ca²⁺ release from intracellular stores but (1) by acting directly on intracellular stores and (2) via activation of a channel different from the IP₃-receptor channel, the so-called ryanodine-sensitive Ca²⁺ channel (17, 31). When cells were exposed to a variety of acrolein concentrations and then subsequently challenged with a low caffeine concentration in order to demonstrate an increase in Ca²⁺ release, we failed to observe any alteration in caffeine-induced Ca²⁺ response. These results support the view that acrolein interferes with receptor-mediated IP₃ signaling, although identification of the precise step(s) in this signaling cascade modulated by acrolein would require additional studies, including alternative experimental approaches.

In our previous studies examining the effect of acrolein on the mechanical response of both rat trachealis and human bronchi to muscarinic stimulation, we observed that this effect was both concentration- and time-dependent. A similar dependence on acrolein concentration and duration of exposure was observed in the present study on calcium signaling. To take into account these two variables, we expressed the change in cholinergic-induced airway responsiveness as a function of product concentration and time (C × T; Figure 5), a surrogate for the dose of acrolein delivered to the tissue. In intact rings, the maximal increase in airway responsiveness occurred for an exposure dose of 6 µM × min in both rat and human preparations (9, 10). Although in the same range, this dose is 3-fold greater than that causing the maximal changes in both [Ca²⁺]_i increase and oscillation frequency in isolated smooth muscle cells (0.2 µM for 10 min, i.e., 2 µM × min). We have re- assessed the effect of acrolein exposure on muscarinic- induced contraction in epithelium-denuded rat tracheal rings. We found that, under these conditions, the effect of acrolein on the maximal response to carbachol was maximal for an intermediate dose, i.e., 3 µM × min. The fact that the maximal increase in Ca²⁺ response occurred in the same range of doses as that in contractile response in epithelial-denuded preparations suggests that the direct effect of this pollutant on calcium signaling may account, at least partially, for acrolein-induced airway hyperresponsiveness.

	Conclusions

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Acrolein increases both the amplitude of the first [Ca²⁺]_i peak induced by a low ACh concentration and the frequency of [Ca²⁺]_i oscillations in response to a high ACh concentration in freshly isolated smooth muscle cells. The effect of acrolein on calcium homeostasis is not observed in response to caffeine, an alternative Ca²⁺ releaser agent in airway smooth muscle. This direct effect of acrolein on calcium homeostasis in airway myocytes may explain, at least partially, the acrolein-induced airway hyperresponsiveness.