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

阐明肌球蛋白磷酸酶在血管内皮细胞和平滑肌细胞中的生理作用

• 批准号: 11670687
• 负责人: HIRANO Katsuya
• 金额: $ 2.3万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670687/
• 关键词: MYOSIN; PROTEIN PHOSPHATASE; REGULATORY SUBUNIT; VASCULAR SMOOTH MUSCLE; VASCULAR ENDOTHELIAL CELL; Ca^<2+> SENSITIVITY; CELL PROLIFERATION; CYTOSKELETON; 血管平滑筋; 血管緊張; 血管構築; MYOSIN; PROTEIN PHOSPHATASE; REGULATORY SUBUNIT; VASCULAR SMOOTH MUSCLE; VASCULAR ENDOTHELIAL CELL; Ca^<2+> SENSITIVITY; CELL PROLIFERATION; CYTOSKELETON; 血管平滑筋; 血管緊張; 血管構築

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

肌球蛋白轻链的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中运动性和细胞骨架组织的调节中也是必不可少的。肌球蛋白磷酸酶最近被克隆，由三个亚基组成。一个38 kDa催化亚基和两个20 kDa和110 kDa的调节亚基。然而，肌球蛋白磷酸酶在血管平滑肌和内皮细胞中的身体作用仍有待研究。在该项目中，我们首先阐明了肌球蛋白磷酸盐在平滑肌收缩中的调节作用，通过检查重组的110 kDa调节型调节型（mypt1）及其竞争群（mypt1）及其ca;在Tritonx100渗透性猪肾动脉条中。构建了MyPT1的一系列截短突变体。将透化纤维与3μmmypt1^<1-633>（片段……更相对应的残留1-633）或mypt1^<39-633>在3 h中孵育ca^<2+>增强了Ca^<2+>诱导的收缩，并导致Ca^<2+> - 张力率ca的左移。在存在180 nm ca^<2+>的情况下，应用3μmmypt1^<1-374>，mypt1^<304-511>或mypt1^<297-374>诱导的收缩，并导致ca^<2+> - 张力曲线的左移。但是，MyPT1^<1-296>缺乏酸性簇对CA^<2+> - 诱导的收缩没有影响。 MyPT1突变体诱导Ca^<2+>的增强与肌球蛋白轻链磷酸化的增加有关。在存在和不存在3μmmypt1^<1-374>的情况下，用300 nm Ca^<2+>获得的肌球蛋白轻链磷酸化水平分别为35.7％和22.4％。 MyPT1^<1-374>将CA^<2+>浓度从10μM变为0 m所引起的弛豫。我们得出的结论是，含有304-374固定的MyPT1的N末端突变体在透明的猪肾动脉中具有CA^<2+>敏化作用。这种作用是由于抑制磷酸酶活性。 304-374保留的区域可能是MyPT1的抑制域。接下来，我们阐明了原位和原发性培养物的内皮细胞表达肌球蛋白磷酸酶的130 kDa亚基，类似于通过抗Mypt1抗体的Western Blot分析的平滑肌MYPT1。在生长的细胞中，MYPT1位于应力纤维上，但是在汇合时，定位模式发生了变化，MyPT1分布在细胞膜附近和细胞 - 细胞接触率附近。对内皮细胞cDNA文库的筛选产生了一个编码89.6 kDa的NH_2末端片段的克隆，与平滑肌mypt1密切相关。建议内皮细胞骨架的调节机制类似于平滑肌收缩的调节机制。较少的