Key Points
- To evaluate the calcium dependence of myosin light chain phosphorylation using cultured tracheal smooth muscle cells as an experimental model.
- Cultured tracheal smooth muscle cells and compared their myosin content and 150-kDa myosin light chain kinase activity to intact tracheal tissue via Western blot analysis.
- Stimulated fura-2-loaded cells with 10 µM carbachol or 10 µM ionomycin to measure free cytosolic calcium concentrations and corresponding myosin light chain phosphorylation.
- Cell stimulation increased cytosolic free calcium from a resting baseline of 165 nM to maximal levels of 4400 nM with ionomycin and 492 nM with carbachol.
- Myosin light chain phosphorylation was triggered when calcium levels exceeded 200 nM, achieving half-maximal phosphorylation (33%) at 260 nM calcium with a Hill coefficient of 2.7.
Structured PICO
PPopulationTracheal smooth muscle cells in culture and tracheal tissue
IInterventionCarbachol (10 microM) or ionomycin (10 microM) stimulation
CComparatorControl (unstimulated cells)
OOutcomeCytosolic free Ca2+ concentration and extent of myosin light chain phosphorylationsurrogate
Small changes in cytosolic Ca2+ concentrations stimulate myosin light chain phosphorylation in smooth muscle cells, suggesting a highly sensitive mechanism for contraction.