Acrolein administered to isolated airways has been shown to alter airway responsiveness as a consequence of its effect on Ca2+ signaling. To examine the mechanisms involved, we studied the effect of acrolein on ACh- and caffeine-induced membrane currents (patch-clamp) in myocytes freshly isolated from rat trachea. In cells clamped at −60 mV, ACh (0.1–10 μM) induced a concentration-dependent inward current, which, in ∼50% of the cells, was followed by current oscillations in response to high concentration of ACh (10 μM). Exposure to acrolein (0.2 μM) for 10 min significantly enhanced the amplitude of the low-ACh (0.1 μM) concentration-induced initial peak of current (318.8 ± 28.3 vs. 251.2 ± 40.3 pA; n = 25,P < 0.05). At a high-ACh concentration (10 μM), the frequency at which subsequent peaks occurred was significantly increased (13.2 ± 1.1 vs. 8.7 ± 2 min−1; n = 20, P < 0.05). ACh-induced current was identified as a Ca2+-activated Cl− current. In contrast, similar exposure to acrolein, which does not alter caffeine-induced Ca2+ release, did not alter caffeine-induced transient membrane currents (595 ± 45 and 640 ± 45 pA in control cells and in cells exposed to acrolein, respectively; n = 15). It is concluded that acrolein alters ACh-induced current as a consequence of its effect on the cytosolic Ca2+ concentration response and that the protective role of inhibitors of Cl− channels in air pollutant-induced airway hyperresponsiveness should be examined.
- cytosolic calcium
- muscarinic stimulation
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). When acrolein or other pollutants are inhaled, airway hyperresponsiveness in a variety of species is induced (1, 7, 12, 13, 18). Information on the health effects of aldehydes is needed because exposure to these pollutants is likely to increase with the use of oxygenated fuel (31).
Our laboratory previously reported that acrolein exposure increases the reactivity of human bronchial and rat tracheal rings to cholinergic agonists in a dose-dependent manner (2, 3). Previous studies have provided indirect evidence that pollutants, among other toxic mechanisms (4), may alter Ca2+ signaling in airway cells (22, 27). We recently obtained direct evidence for such an effect of acrolein in isolated rat tracheal smooth muscle cells (28). We have observed that acrolein increases the amplitude of the first cytosolic Ca2+concentration ([Ca2+]i) peak and the frequency of subsequent [Ca2+]i oscillations in response to low and high acetylcholine (ACh) concentrations, respectively, for a range of “doses” of acrolein (estimated as the product of the concentration of acrolein by the duration of exposure) similar to that for increasing isometric contractions (8,28).
So far, the effect of pollutants on ion channels in smooth muscle has received very little attention. It has been observed that ozone (O3) does not modify contractile responses to KCl in dog tracheal smooth muscle, indirectly suggesting that this pollutant has no effect on voltage-dependent Ca2+ channels (13). We obtained similar indirect evidence with acrolein (3). However, to the best of our knowledge, the effect of air pollutants on agonist-induced membrane current has not been examined. Therefore, the aim of the present study was to study the effect of acrolein on ACh- and caffeine-induced membrane currents in airway myocytes.
MATERIALS AND METHODS
Rat tracheae were obtained from 10- to 15-wk-old male Wistar rats weighing 300–400 g. For each experiment, a rat was anesthetized by intraperitoneal administration of 400 mg of ethyl carbamate. Heart and lungs were removed en bloc, and the trachea was rapidly dissected out. The muscular strip located on the dorsal face of the trachea was further dissected under binocular control, as previously described (8, 28). 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 in low-Ca2+ PSS containing 1.0 mg/ml collagenase, 0.7 mg/ml pronase, 0.06 mg/ml elastase, and 3 mg/ml bovine serum albumin 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. To attach onto glass coverslips, cells were stored for 1–3 h at 4°C in PSS containing 0.8 mM Ca2+; they were used on the same day.
Membrane current recordings.
Membrane currents were measured, as previously described (6), in conventional whole cell current recording mode using a Biologic RK 400 patch-clamp amplifier. Whole cell membrane currents were recorded with borosilicate patch pipettes of 3- to 7-MΩ resistance obtained with a vertical puller (Narishige, Tokyo, Japan). Membrane currents were stored and analyzed using an IBM-PC computer (pCLAMP system, Axon Instruments, Foster City, CA). The currents were not corrected for leakage. Currents were recorded under a constant voltage clamp at −60 mV and during a ramp pulse (0.5 s in duration, 0.2 V/s) from −60 to + 40 mV. Current-voltage (I-V) relation for ACh- or caffeine-induced current was obtained by subtracting current evoked by the same ramp pulse in the absence of agonist.
Exposure to acrolein.
Exposure of isolated cells to acrolein was performed by immersing the coverslips with attached cells for 10 min in PSS containing an acrolein concentration of 0.2 μM. 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.
Solutions and application of agonists.
The external PSS contained (in mM) 130 NaCl, 5.6 KCl, 1 MgCl2, 2 CaCl2, 11.1 d-glucose, and 10 HEPES, pH 7.4 with NaOH. Ca2+-free PSS was prepared by replacing CaCl2 with 0.4 mM EGTA.
The internal solution (the solution in the patch pipette and inside the cell) contained (in mM) 120 CsCl, 5 NaCl, 20 HEPES, 1 MgCl2, and 2.5 Na2ATP, pH 7.1 with NaOH.
Agonists were applied to the recorded cell by pressure ejection from a glass pipette located close to the cell for the period indicated on the records. It was verified, in control experiments, that no change in membrane current was observed during test ejection of PSS. Experiments were done at room temperature (20–22°C).
Collagenase (type CLS1) was from Worthington Biochemical (Freehold, NJ). Acrolein, minimum 90% pure and stabilized with 0.1% hydroquinone, pronase (type E), elastase (type 3), bovine serum albumin, ACh, and caffeine were purchased from Sigma Chemical (Saint Quentin Fallavier, France).
Data analysis and statistics.
Results of membrane current are expressed as means ± SE, withn equal to the number of the 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 pairedt-tests.
Results were considered significant at P < 0.05.
Characterization of ACh-induced membrane current in isolated tracheal myocytes.
In this series of experiments, we assessed the effect of ejection of ACh (30 s) on membrane current in cells clamped at −60 mV, a value close to that of the resting potential in this type of smooth muscle (21). ACh (0.1–10 μM) induced a concentration-dependent inward current, the amplitude of which increased from 185.3 ± 29 to 452 ± 32 pA (n= 15–50) (Fig. 1 A). TheI-V relation, obtained by applying a ramp pulse from −60 to + 40 mV at the peak of the current, was mainly linear, and the reversal potential (E rev = 4.9 ± 0.8 mV; n = 8) was close to the equilibrium potential for Cl− (E Cl = −2.6 mV in our experimental conditions) (Fig. 1 B). Pretreatment of cells held at −60 mV with niflumic acid (10–50 μM) for 10 min reduced the amplitude of the ACh-induced current in a concentration-dependent manner (Fig. 1 C). In ∼50% of the cells, when the ACh concentration increased, the first peak current was followed by current oscillations of decreasing amplitude (Fig.1 A).
Effect of acrolein exposure on ACh-induced membrane current in isolated tracheal myocytes.
Exposure to acrolein (0.2 μM for 10 min) significantly enhanced the amplitude of the low-ACh concentration-induced initial peak of current (318.8 ± 28.3 vs. 251.2 ± 40.3 pA;n = 4, P < 0.05, Fig.2). For a high-ACh concentration (10 μM), the value of the first peak current was not changed by acrolein exposure, but the frequency at which subsequent peaks occurred was significantly increased (13.2 ± 1.1 vs. 8.7 ± 2 min−1; n = 4, P < 0.05, Fig. 2 Ab).
Effect of acrolein exposure on caffeine-induced membrane current in isolated tracheal myocytes.
In cells clamped at −60 mV, application of caffeine (5 mM for 5 s) induced only one large transient peak of inward current, the amplitude of which was 595 ± 45 pA (n = 15) (Fig.2 C). The I-V relation, obtained by applying a ramp pulse from −60 to +40 mV at the peak of the single caffeine-induced current, was mainly linear and similar to that for ACh-induced current. The E rev was −3.8 mV (n = 4). Exposure to acrolein (0.2 μM for 10 min) did not alter caffeine-induced transient membrane current (640 ± 45 pA; n = 15, P > 0.05, Fig.2 C).
The present study indicates that exposure of freshly isolated airway myocytes to acrolein alters ACh-induced membrane current solely as a consequence of its effect on the [Ca2+]iresponse.
In the present study, we have investigated the effect of a single concentration of acrolein (0.2 μM) administered for a fixed duration of exposure (10 min) on the basis of our previous studies in isolated cells (8, 28). As previously discussed (2,8), this dose is lower than that required to produce airway hyperresponsiveness in live animals (17, 18) and therefore relevant to the investigation of the cellular mechanisms implicated in acrolein-induced effects.
In agreement with previous reports on airway smooth muscle from a variety of species including dog (10), horse (5), guinea pig (11), cat (32), or human (9), we have observed, in this study, that ACh, as well as caffeine, activates a membrane current that corresponds to a Ca2+-activated Cl− current [I Cl(Ca)] in rat tracheal smooth muscle cells. Identification of the nature of this membrane current is based on the following reasons. First, in our experimental conditions, that is, E Cl of −2.6 mV and holding potential of −60 mV, the current was inwardly directed as expected from the large outward electrochemical gradient for Cl−. Second, in control conditions, its E rev was close to the theoretical E Cl . Third, it was inhibited in a concentration-dependent manner by niflumic acid, a potent inhibitor of agonist-induced Cl− currents in smooth muscle (16). Complete blockade was obtained for a relatively low concentration (50 μM). Up to 100 μM, niflumic acid is selective for Cl− currents in airway smooth muscle cells (9). Finally, this current became an oscillatory inward membrane current in response to high concentrations of ACh, which trigger [Ca2+]i oscillations in this preparation (28), whereas caffeine, which elicits single transient [Ca2+]i rises (28), also elicited a transient inward Cl− current that does not oscillate.
The effect of acrolein on ACh-induced membrane current thus appears to be a consequence of its sole effect on the [Ca2+]i response. Indeed, acrolein increased the amplitude and the frequency of oscillation of the current elicited by low and high concentrations of ACh, respectively, an effect identical to that on ACh-induced [Ca2+]iresponse (8, 28). Because our laboratory previously demonstrated, in pulmonary vascular smooth muscle cells, that the amplitude of agonist-induced I Cl(Ca) was related to that of the increase in [Ca2+]i produced by the same agonist (6), it is suggested that that the effect of acrolein on ACh-induced membrane current is indirect. This suggestion is supported by the fact that, in contrast, similar exposure to acrolein, which does not alter caffeine-induced Ca2+release (28), also did not alter the caffeine-induced transient membrane current. Nevertheless, the acrolein-induced increase in I Cl(Ca) may, in turn, promote membrane depolarization, thus contributing to the increase in tone (9, 10,29). Therefore, the protective role of inhibitors of Cl− channels in air pollutant-induced airway hyperresponsiveness should be examined.
The effect of acrolein on the oscillatoryI Cl(Ca) as a consequence of its effect on [Ca2+]i oscillations deserves further discussion. At the whole cell level, cholinergic-induced [Ca2+]i oscillations in airway smooth muscle cells are primarily inositol trisphosphate dependent, involve a cyclic Ca2+ release-Ca2+ reuptake by intracellular store, and have frequency increases with increases in cholinergic agonist concentration (19, 20, 23, 29). In the present study, we have observed that, as for the first [Ca2+]i peak and [Ca2+]i oscillation frequency (8,28), acrolein increases the first transient current and the frequency of subsequent current oscillations. Such an increase in oscillation frequency is likely to be responsible for the acrolein-induced effect on the mechanical activity, since an increase in airway tone depends on an increase in Ca2+ oscillation frequency (28, 29). The use of real-time confocal imaging has provided new information on the mechanisms underlying Ca2+ oscillations at both the subcellular and whole cell level (14, 15, 24-26, 30). ACh-induced propagating oscillations of [Ca2+]i have been described in airway smooth muscle cells as the result from repetitive release of Ca2+ through ryanodine-receptor (RyR) channels (14,30). Spontaneous localized Ca2+ transients (Ca2+ sparks), which represent unitary Ca2+release through RyR channels, have been described in airway smooth muscle cells (30), and it has been demonstrated that ACh-induced [Ca2+]i oscillations represent a spatial and temporal integration of Ca2+ sparks (24). The effect of increasing ACh concentration is different at the subcellular vs. the whole cell level. At the subcellular level, the amplitude of regional [Ca2+]i oscillations remains constant when ACh concentration increases, whereas frequency and propagation velocity increase. At the whole cell level, global [Ca2+]i oscillations appear as a concentration-dependent increase in both peak and mean cellular [Ca2+]i (25). In the present study, acrolein increased both peak and oscillation frequency of the whole cell ACh-induced membrane current as a consequence of its effect on global [Ca2+]i oscillations. The subcellular effect of this pollutant on both Ca2+sparks and spatial and temporal integration of such sparks requires further examination.
We thank Dr. E. Roux for helpful advice in establishing the cell acrolein exposure protocol.
This work was supported by grants from the Ministère de l'Environnement and Agence De l'Environnement Et de la Maı̂trise d'Energie (ADEME; no. 9962035), Conseil Régional d'Aquitaine (no. 980301115), and Mutuelle Générale de l'Education Nationale.
J. M. Hyvelin was supported by a doctoral scholarship from ADEME.
Address for reprint requests and other correspondence: R. Marthan, Laboratoire de Physiologie Cellulaire Respiratoire, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France (E-mail:).
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- Copyright © 2001 the American Physiological Society