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

Abstract

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.

Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation

“…For ex vivo imaging of lung microvessels, lungs were inflated with 1.5 ml of 2% agarose-HBSS at 37°C, and lungs were stiffened at 4°C as described previously (72). The stiffened lung tissue was placed in EMS-5000 tissue slicer (Electron Microscopy Sciences) and approximately 200-μm slices were used for Ca 2+ imaging.…”

Ca2+-sensing Transgenic Mice

“…Images showing cross-sections of the arterial walls were captured through the center, parallel to the long axis, of an intact pressurized (70 mm Hg) mesenteric artery at 35°C. The lung slice preparation was similar to that previously described (9). Briefly, the trachea was cannulated and the lungs filled with 2% agarose (type VII-A: low gelling temperature) before removal.…”

Role of Protein Kinase Network in Excitation-Contraction Coupling in Smooth Muscle Cell

“…In human bronchial smooth muscle, for example, RyR, though present and functional, do not participate in the acetylcholine-induced Ca 2+ response (Hyvelin et al, 2000a). (Gunter et al, 2000;Roux et al, 2004 (Bergner et al, 2002;Hyvelin et al, 2000b;Kajita et al, 1993;Liu et al, 1996). Changes in [Ca 2+ ] i are not uniform within the cytosol, and studies have evidenced the role of local change in Ca 2+ signalling (Prakash et al, 2000).…”