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 (similar to75-mum 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 air- way lumen and a transient increase in [Ca2+], in individual SMCs that subsequently declined to initiate multiple intracellular Ca2+ oscillations. These Ca2+ oscillations spread as Ca2+ waves through the SMCs at similar to48 mum/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 BATIA 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 airways The concentration dependence of the airway contraction matched the concentration dependeuce 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 IPs and ryanodine receptors, and are required to maintain airway contraction.