Ehrlich H P, Rajaratnam J B, Griswold T R
J Cell Physiol. 1986 Aug;128(2):223-30. doi: 10.1002/jcp.1041280213.
When 1 mM ATP is added to human dermal fibroblasts (DF) in monolayer culture permeabilized by glycerol, they undergo a rapid reduction in length and their intracellular actin filaments aggregate. This process is referred to as cell contraction. Treating glycerol-permeabilized DF with alkaline phosphatase before adding 1 mM ATP should cause dephosphorylation. Dephosphorylated preparations do not undergo cell contraction initiated by ATP. When myosin light-chain kinase (MLCK) isolated from turkey gizzard is added with cofactors to cells dephosphorylated by alkaline phosphatase treatment, contraction is restored. DF incubated for 24 h with db cAMP or cholera toxin show elevated intracellular concentrations of cAMP and little cell contraction. Contraction is reestablished when MLCK with cofactors is incubated with these preparations before ATP is added. Fibroblasts from Epidermolysis Bullosa dystrophica recessive patients produce excess cAMP. Those cells show minimal contraction, however; treating them with MLCK and cofactors renews contraction brought about by ATP. When DF are incubated with trifluoperazine to block calmodulin-dependent enzyme reactions, cell contraction is inhibited. Adding cytochalasin B disrupts microfilaments and also inhibits contraction. This work supports the idea that myosin ATPase is critical to cell contraction. Myosin ATPase is dependent on the phosphorylation of the regulatory peptide, myosin light chain. Elevating intracellular concentrations of cAMP or treatment of permeabilized cell preparations with alkaline phosphatase may inhibit myosin ATPase activity. The restoration of phosphorylation by adding MLCK with cofactors served to reestablish cell contraction.
当向经甘油通透处理的单层培养的人皮肤成纤维细胞（DF）中加入1 mM ATP时，它们会迅速缩短长度，并且细胞内的肌动蛋白丝会聚集。这个过程被称为细胞收缩。在加入1 mM ATP之前，用碱性磷酸酶处理经甘油通透处理的DF会导致去磷酸化。去磷酸化的制剂不会发生由ATP引发的细胞收缩。当将从火鸡砂囊中分离出的肌球蛋白轻链激酶（MLCK）与辅因子一起加入经碱性磷酸酶处理去磷酸化的细胞时，收缩得以恢复。用二丁酰环磷腺苷（db cAMP）或霍乱毒素孵育24小时的DF显示细胞内cAMP浓度升高且几乎没有细胞收缩。在加入ATP之前，将MLCK与辅因子一起孵育这些制剂时，收缩得以重新建立。隐性营养不良型大疱性表皮松解症患者的成纤维细胞会产生过量的cAMP。然而，这些细胞显示出最小程度的收缩；用MLCK和辅因子处理它们会恢复由ATP引起的收缩。当DF与三氟拉嗪一起孵育以阻断钙调蛋白依赖性酶反应时，细胞收缩受到抑制。加入细胞松弛素B会破坏微丝并也抑制收缩。这项工作支持了肌球蛋白ATP酶对细胞收缩至关重要的观点。肌球蛋白ATP酶依赖于调节肽肌球蛋白轻链的磷酸化。提高细胞内cAMP浓度或用碱性磷酸酶处理通透的细胞制剂可能会抑制肌球蛋白ATP酶活性。通过加入MLCK与辅因子恢复磷酸化作用可重新建立细胞收缩。
