2000 Fiscal Year Final Research Report Summary
Elucidation of the physiological role of myosin phosphatase in vascular endothelial cells and smooth muscle cells
| Project/Area Number |
11670687
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Circulatory organs internal medicine
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
HIRANO Katsuya GRAD SCH MED, SCI.KYUSHU UNIVERSITY LECTURER, 大学院・医学研究院, 講師 (80291516)
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| Co-Investigator(Kenkyū-buntansha) |
JUNJI Nishimura GRAD SCH MED SCI, KYUSHU UNIVERSITY ASCOSSIATE PROF., 大学院・医学研究院, 助教授 (90237727)
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| Project Period (FY) |
1999 – 2000
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| Keywords | MYOSIN / PROTEIN PHOSPHATASE / REGULATORY SUBUNIT / VASCULAR SMOOTH MUSCLE / VASCULAR ENDOTHELIAL CELL / Ca^<2+> SENSITIVITY / CELL PROLIFERATION / CYTOSKELETON |
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| Research Abstract |
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
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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
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