Taylor D A, Stull J T
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1988 Oct 5;263(28):14456-62.
Smooth muscle cells grown in culture may provide a model system for studying the Ca2+ dependence of myosin light chain phosphorylation. Tracheal smooth muscle cells in culture had 60% of the myosin content of tracheal tissue. Western analysis with appropriate antibodies demonstrated one 20-kDa light chain and the presence of a 150-kDa myosin light chain kinase in both tracheal smooth muscle tissue and cells. Moreover, tracheal cells contained 74% of the myosin light chain kinase activity measured in tissue. Similar types of analyses of nonmuscle cells showed a much lower myosin and myosin light chain kinase content. Carbachol (10 microM) or ionomycin (10 microM) stimulation of fura-2-containing cells resulted in a rapid increase in cytosolic free Ca2+ concentration and in the extent of myosin light chain phosphorylation. Maximal increases in Ca2+ concentrations were greater with ionomycin than with carbachol (4400 versus 492 nM). Light chain phosphorylation increased after the Ca2+ concentration exceeded 200 nM from control values of 165 nM. Half-maximal phosphorylation (33%) occurred at 260 nM Ca2+. There was a similar relationship between free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation in carbachol- and ionomycin-stimulated cells. This relationship had a Hill coefficient of 2.7. These observations indicate that small changes in Ca2+ concentrations stimulate myosin light chain phosphorylation and thus presumably contraction in smooth muscle cells.
在培养中生长的平滑肌细胞可能为研究肌球蛋白轻链磷酸化对钙离子的依赖性提供一个模型系统。培养的气管平滑肌细胞的肌球蛋白含量为气管组织的60%。用适当抗体进行的蛋白质免疫印迹分析表明，气管平滑肌组织和细胞中均存在一条20 kDa的轻链以及150 kDa的肌球蛋白轻链激酶。此外，气管细胞中的肌球蛋白轻链激酶活性为组织中所测活性的74%。对非肌肉细胞进行的类似分析显示，其肌球蛋白和肌球蛋白轻链激酶含量要低得多。用卡巴胆碱（10 μM）或离子霉素（10 μM）刺激含fura-2的细胞，可导致胞质游离钙离子浓度迅速升高以及肌球蛋白轻链磷酸化程度增加。离子霉素引起的钙离子浓度最大增幅大于卡巴胆碱（4400对492 nM）。当钙离子浓度从对照值165 nM超过200 nM后，轻链磷酸化增加。半最大磷酸化（33%）发生在钙离子浓度为260 nM时。在卡巴胆碱和离子霉素刺激的细胞中，胞质游离钙离子浓度与肌球蛋白轻链磷酸化程度之间存在类似关系。这种关系的希尔系数为2.7。这些观察结果表明，钙离子浓度的微小变化会刺激肌球蛋白轻链磷酸化，从而可能引起平滑肌细胞收缩。
