Elucidation of the physiological role of myosin phosphatase in vascular endothelial cells and smooth muscle cells

肌球蛋白轻链磷酸化在血管张力调节中起关键作用。此外，肌球蛋白磷酸化在非肌肉细胞的运动和细胞骨架组织的调节中也是必不可少的。肌球蛋白磷酸酶是最近克隆的，由三个亚基组成；一个38 kDa催化亚基和两个20 kDa和110 kDa调节亚基。然而，肌球蛋白磷酸酶在血管平滑肌和内皮细胞中的生理作用仍有待研究。在本项目中，我们首先通过检测重组110 kDa调控亚基（MYPT1）及其突变体对tritonx100渗透猪肾动脉条Ca^<2+>-诱导的收缩和肌球蛋白轻链磷酸化的影响，阐明了肌球蛋白磷酸酯在平滑肌收缩中的调节作用。构建了一系列MYPT1的截断突变体。与3 μM MYPT1^<1-633>（一个片段…更多对应残基1-633）或MYPT1^<39-633>孵育3 h后，Ca^<2+>诱导收缩增强，Ca^<2+>-张力曲线左移。应用3 μM MYPT1^<1-374>、MYPT1^<304-511>或MYPT1^<297-374>，在180 nM Ca^<2+>存在下，引起Ca^<2+>-张力曲线左移。然而，缺乏酸性簇的MYPT1^<1-296>对Ca^<2+>诱导的收缩没有影响。MYPT1突变体增强Ca^<2+>-诱导的收缩与肌球蛋白轻链磷酸化的增加有关。在3 μM MYPT1^<1-374>存在和不存在时，300 nM Ca^<2+>的肌球蛋白轻链磷酸化水平分别为35.7%和22.4%。将Ca^<2+>浓度从10 μM改变到0 μM时，MYPT1^<1-374>延缓了弛豫。我们得出结论，含有304-374残基的MYPT1 n端突变体在通透性猪肾动脉中具有Ca^<2+>增敏作用。这种作用是由于抑制了磷酸酶的活性。304-374个残基区域可能是MYPT1的抑制结构域。接下来，我们通过抗MYPT1抗体的western blot分析，阐明了内皮细胞在原位和原代培养中表达130 kDa的肌球蛋白磷酸酶亚基，与平滑肌MYPT1相似。在生长的细胞中，MYPT1定位在应力纤维上，但在汇合时，定位模式改变，MYPT1分布在靠近细胞膜和细胞间接触处。内皮细胞cDNA文库筛选得到一个编码89.6 kDa的nh_2末端片段的克隆，该片段与平滑肌MYPT1密切相关。内皮细胞骨架的调节机制与平滑肌收缩的调节机制相似。少

The phosphorylation of myosin light chain plays a key role in the regulation of vascular tone. In addition, myosin phosphorylation is also essential in the regulation of motility and cytoskeletal organization in the non-muscle cells. The myosin phosphatase has been recently cloned and is composed of three subunits ; a 38 kDa catalytic subunit and two regulatory subunits of 20 kDa and 110 kDa. However, the physiological role of myosin phosphatase in the vascular smooth muscle and endothelial cells remained to be investigated.In this project, we first elucidated the regulatory role of the myosin phosphates in smooth muscle contraction by examining the effects of the recombinant 110 kDa regulatory subunit (MYPT1) and its mutants on the Ca^<2+>-induced contraction and myosin light chain phosphorylation in the TritonX100-permeabilized porcine renal arterial strips. Series of truncation mutants of MYPT1 were constructed. Incubating the permeabilized fibers with 3 μM MYPT1^<1-633> (a fragment … More corresponding to residues 1-633) or MYPT1^<39-633> for 3 h enhanced the Ca^<2+> induced contraction and caused a leftward shift of the Ca^<2+>-tension curve. Application of 3 μM MYPT1^<1-374>, MYPT1^<304-511> or MYPT1^<297-374> induced contractions in the presence of 180 nM Ca^<2+> and caused a leftward shift of the Ca^<2+>-tension curve. However, MYPT1^<1-296> lacking an acidic cluster had no effect on the Ca^<2+>-induced contraction. The enhancement of Ca^<2+>-induced contraction by MYPT1 mutants was associated with an increase in myosin light chain phosphorylation. The level of myosin light chain phosphorylation obtained with 300 nM Ca^<2+> in the presence and absence of 3 μM MYPT1^<1-374> were 35.7 % and 22.4 %, respectively. The relaxation induced by changing Ca^<2+> concentraion from 10 μM to 0 M was retarded by MYPT1^<1-374>. We concluded that the N-terminal mutants of MYPT1 containing the 304-374 residues had a Ca^<2+> sensitizing effect in permeabilized porcine renal artery. This effect is due to inhibition of phosphatase activity. The region of 304-374 residues may be an inhibitory domain of MYPT1.We next elucidated that endothelial cells, in situ and in primary culture, express the 130 kDa subunit of myosin phosphatase, similar to the smooth muscle MYPT1 by the western blot analysis with anti-MYPT1 antibody. In the growing cells, MYPT1 was localized on stress fiber, but at confluence the localization pattern changed and MYPT1 was distributed close to the cell membrane and at cell-cell contacts. Screening of an endothelial cell cDNA library yielded a clone encoding an NH_2-terminal fragment of 89.6 kDa, closely related to smooth muscle MYPT1. The regulatory mechanism of endothelial cytoskeleton was suggested to be similar to the regulatory mechanism of smooth muscle contraction. Less