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

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的调节中起关键作用。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。肌球蛋白磷酸酶最近已被克隆，由三个亚基组成：一个38 kDa的催化亚基和两个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生理作用仍有待研究。我们首先通过检测重组的110 kDa调节亚基（MYPT 1）及其突变体对TritonX 100细胞中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化在平滑肌收缩中的调节作用。透化猪肾动脉条。构建了MYPT 1的一系列截短突变体。用3 μM MYPT 1 ^（片段）孵育透化纤维<1-633> ...更多信息 与MYPT 1 ^<39-633>作用3 h后，Ca^2+-张力曲线发生明显的位移，并与MYPT 1 ^作用3 h后，Ca^2+-张力曲线发生明显的位移。在180 nM Ca^2+存在的情况下，应用3 μM MYPT 1 ^<1-374>、MYPT 1 ^<304-511>或MYPT 1 ^可<297-374>诱导收缩，并引起Ca^2+-张力曲线的显著移动。然而，缺乏酸性簇的MYPT 1 ^<1-296>对Ca^&lt;2+&gt;诱导的收缩没有影响。MYPT 1突变体对Ca^&lt;2+&gt;诱导的收缩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3 μM MYPT 1 ^的情况下，用300 nM Ca^&lt;2+&gt;获得的肌球蛋白轻链磷酸化水平<1-374>分别为35.7%和22.4%。MYPT 1 ^可抑制Ca^&lt;2+&gt;浓度从10 μM到0 M引起的舒张<1-374>。我们的结论是，MYPT 1的N端突变体（包含304-374个残基）在透化的猪肾动脉中具有Ca^2+增敏作用。这种作用是由于磷酸酶活性的抑制。304-374位残基的区域可能是MYPT 1的抑制结构域。用抗MYPT 1抗体进行的Western blot分析表明，在原代培养和原位培养的内皮细胞表达与平滑肌MYPT 1相似的肌球蛋白磷酸酶130 kDa亚基。在生长的细胞中，MYPT 1位于应力纤维上，但在汇合时，定位模式发生变化，MYPT 1分布在细胞膜附近和细胞与细胞接触处。从内皮细胞cDNA文库中筛选得到一个编码与平滑肌MYPT 1密切相关的89.6kDa的NH_2端片段的克隆。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

项目成果

Hirano M, Niiro N, Hirano K et al.: "Expression, subcellular localization, and cloning of the 130-kDa regulatory subunit of myosin phosphatase in porcine aortic endothelial cells"Biochemical and Biophysical Research Communications. 254. 490-496 (1999)

Hirano M、Niiro N、Hirano K 等人：“猪主动脉内皮细胞中肌球蛋白磷酸酶 130-kDa 调节亚基的表达、亚细胞定位和克隆”生物化学和生物物理研究通讯。

Hartshorne DJ,Hirano K: "Interactions of protein phosphatase type1, with a focus on myosin phosphatase."Molecular and Cellular Biochemistry. 190. 79-84 (1999)

Hartshorne DJ、Hirano K：“1 型蛋白磷酸酶的相互作用，重点是肌​​球蛋白磷酸酶。”分子和细胞生物化学。

Hartshorne DJ,Hirano K: "Interactions of protein phosphatase type 1, with a focus on myosin phosphatase."Molecular and Cellular Biochemistry. 190. 79-84 (1999)

Hartshorne DJ、Hirano K：“1 型蛋白磷酸酶的相互作用，重点是肌​​球蛋白磷酸酶。”分子和细胞生物化学。

Hirano M,Niiro N,Hirano K,Nishimura J,Hartshorne DJ,Kanaide H: "Expression, subcellular localization and cloning of the 130 kDa regulatory subunit of myosin phosphatase in porcine aortic endothelial cells."Biochemical and Biophysical Research Communicatio

Hirano M、Niiro N、Hirano K、Nishimura J、Hartshorne DJ、Kanaide H：“猪主动脉内皮细胞中肌球蛋白磷酸酶 130 kDa 调节亚基的表达、亚细胞定位和克隆。”生物化学和生物物理研究通讯

Y.B.Zhou, K.Hirano, C.Sakihara, J.Nishimura, H.Kanaide.: "NH_2-terminal fragments of 130 kDa subunit of myosin phosphatase increases Ca^<2+> sensitivity of the porcine renal artery."Journal of Physiology. vol.516. 55-65 (1999)

Y.B.Zhou、K.Hirano、C.Sakihara、J.Nishimura、H.Kanaide.：“肌球蛋白磷酸酶130kDa亚基的NH_2末端片段增加猪肾动脉的Ca^2敏感性。”生理学杂志。