Acetylcholine-induced Calcium Signaling and Contraction of Airway Smooth Muscle Cells in Lung Slices

The Ca²⁺ signaling and contractility of airway smooth muscle cells (SMCs) were investigated with confocal microscopy in murine lung slices (∼75-μm thick) that maintained the in situ organization of the airways and the contractility of the SMCs for at least 5 d. 10–500 nM acetylcholine (ACH) induced a contraction of the airway lumen and a transient increase in [Ca²⁺]_i in individual SMCs that subsequently declined to initiate multiple intracellular Ca²⁺ oscillations. These Ca²⁺ oscillations spread as Ca²⁺ waves through the SMCs at ∼48 μm/s. The magnitude of the airway contraction, the initial Ca²⁺ transient, and the frequency of the subsequent Ca²⁺ oscillations were all concentration-dependent. In a Ca²⁺-free solution, ACH induced a similar Ca²⁺ response, except that the Ca²⁺ oscillations ceased after 1–1.5 min. Incubation with thapsigargin, xestospongin, or ryanodine inhibited the ACH-induced Ca²⁺ signaling. A comparison of airway contraction with the ACH-induced Ca²⁺ response of the SMCs revealed that the onset of airway contraction correlated with the initial Ca²⁺ transient, and that sustained airway contraction correlated with the occurrence of the Ca²⁺ oscillations. Buffering intracellular Ca²⁺ with BAPTA prohibited Ca²⁺ signaling and airway contraction, indicating a Ca²⁺-dependent pathway. Cessation of the Ca²⁺ oscillations, induced by ACH-esterase, halothane, or the absence of extracellular Ca²⁺ resulted in a relaxation of the airway. The concentration dependence of the airway contraction matched the concentration dependence of the increased frequency of the Ca²⁺ oscillations. These results indicate that Ca²⁺ oscillations, induced by ACH in murine bronchial SMCs, are generated by Ca²⁺ release from the SR involving IP₃- and ryanodine receptors, and are required to maintain airway contraction.