Wysolmerski R B, Lagunoff D
Department of Pathology, Saint Louis University School of Medicine, MO 63104.
Proc Natl Acad Sci U S A. 1990 Jan;87(1):16-20. doi: 10.1073/pnas.87.1.16.
Permeabilized bovine pulmonary artery endothelial cell monolayers were used to investigate the mechanism of endothelial cell retraction. Postconfluent endothelial cells permeabilized with saponin retracted upon exposure to ATP and Ca2+. Retraction was accompanied by thiophosphorylation of 19,000-Da myosin light chains when adenosine 5'-[gamma-[35S]thio]triphosphate was included in the medium. Both retraction and thiophosphorylation of myosin light chains exhibited a graded quantitative dependence on Ca2+. When permeabilized monolayers were extracted in buffer D containing 100 mM KCl and 30 mM MgCl2 for 30 min, the cells failed to retract upon exposure to ATP and Ca2+, and no thiophosphorylation of myosin light chains occurred. The ability both to retract and to thiophosphorylate myosin light chains was restored by the addition to the permeabilized, extracted cells of myosin light-chain kinase and calmodulin together but not by either alone. These studies indicate that endothelial cell retraction, as does smooth muscle contraction, depends on myosin light-chain kinase phosphorylation of myosin light chains.
使用通透化的牛肺动脉内皮细胞单层来研究内皮细胞回缩的机制。用皂角苷通透化的汇合后内皮细胞在暴露于ATP和Ca2+时会回缩。当培养基中包含腺苷5'-[γ-[35S]硫代]三磷酸时，回缩伴随着19,000道尔顿肌球蛋白轻链的硫代磷酸化。肌球蛋白轻链的回缩和硫代磷酸化均对Ca2+表现出分级定量依赖性。当通透化的单层细胞在含有100 mM KCl和30 mM MgCl2的缓冲液D中提取30分钟后，细胞在暴露于ATP和Ca2+时不会回缩，并且肌球蛋白轻链也不会发生硫代磷酸化。通过将肌球蛋白轻链激酶和钙调蛋白一起添加到通透化、提取后的细胞中可恢复肌球蛋白轻链的回缩和硫代磷酸化能力，但单独添加其中任何一种均无此效果。这些研究表明，内皮细胞回缩与平滑肌收缩一样，取决于肌球蛋白轻链激酶对肌球蛋白轻链的磷酸化作用。
