We have previously observed that acrolein administered ex vivo to isolated airways alters the subsequent airway responsiveness. To examine the cellular mechanisms involved in this alteration, we have studied the effect of acrolein exposure on calcium signaling in myocytes freshly isolated from rat trachea. We have also studied the effect of acrolein exposure on isometric contraction of rat epithelium-free tracheal rings. Tissues were exposed to a variety of acrolein concentrations from 0.1 to 1 microM and durations from 5 to 15 min. In isolated cells, exposure to acrolein did not modify the resting cytosolic Ca2+ concentration ([Ca2+]i) whatever the concentration or duration of exposure, but altered the pattern of the Ca2+ response to acetylcholine (ACh). ACh typically induces an initial [Ca2+]i rise followed by peaks of decreasing amplitude (oscillations). Exposure to a fixed concentration of acrolein (0.2 microM) for 5 and 10 min significantly enhanced the amplitude of the initial [Ca2+]i rise in response to a low concentration of ACh (0.1 microM) by 50.8 and 77%, respectively. Similarly, exposure for a fixed duration of 10 min significantly enhanced the amplitude of the initial [Ca2+]i rise by 49.4% at an acrolein concentration of 0.3 microM. When cells were stimulated with a high ACh concentration (10 microM), the value of the first [Ca2+]i peak was not changed by acrolein exposure; but the frequency at which subsequent peaks occurred was significantly increased by 44.4% after 10 min of exposure to a fixed concentration of 0.2 microM and by 36.3% following an exposure for a fixed duration of 10 min at the concentration of 0.3 microM. In contrast, acrolein, whatever the concentration, had no effect on the caffeine-induced [Ca2+]i response. In rat epithelium-free tracheal rings, acrolein increased the response to muscarinic stimulation, with a maximal effect observed for an exposure to 0.3 microM for 10 min. The effect of acrolein on the [Ca2+]i response of isolated myocytes occurred over a range of doses similar to that on the contractile response of rings, suggesting that the effect of this pollutant on calcium signaling may account, at least partially, for acrolein-induced airway hyperresponsiveness.