1997 Fiscal Year Final Research Report Summary
Intracellular Signal Transduction and Cytoskeleton in Endothelial Cells
| Project/Area Number |
07670772
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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 | Hamamatsu University School of Medicine |
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Principal Investigator |
WATANABE Hiroshi Hamamatsu University School of Medicine, Internal Medicine III,Research Associate, 医学部附属病院, 助手 (50262803)
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| Co-Investigator(Kenkyū-buntansha) |
TERAKAWA Susumu Hamamatsu University School of Medicine, Photon Medical Research Cent r, Profess, 医学部光量子医学研究センター, 教授 (50014246)
HAYASHI Hideharu Hamamatsu University School of Medicine, Photon Medical Research Center, Assista, 医学部光量子医学研究センター, 助教授 (50135258)
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| Project Period (FY) |
1995 – 1997
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| Keywords | Endothelial Cells / Calcium / Myosin Light-Chain Kinase / Bradykinin / Thapsigargin / Fluid flow / Myosin Light-Chain |
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| Research Abstract |
Cytosolic Ca^<2+> ([Ca^<2+>]i) plays an important role in endothelial cell signaling. Although it has been suggested that the influx of Ca^<2+>can be triggered by depletion of intracellular Ca^<2+>stores, the mechanism (s) underlying this phenomenon needs further elaboration. In the present study, involvement of myosin light-chain kinase (MLCK) in the regulation of Ca^<2+>-signailing was investigated in agonist-and fluid-flow-stimulated endothelial cells loaded with Ca^<2+>sensitive dyes.
Bradykinin (BK) and thapsigargin caused an increase in [Ca^<2+>]_i followed by a sustained rise due to Ca^<2+>influx from extracellular space and shifted total myosin light-chain (MLC) from the unphosphorylated to the diphosphorylated from. ML-9 (100muM), an inhibitor of MLCK,abolished the Ca^<2+>influx and prevented MLC diphosphorylation in BK-and thapsigargin-treated cells, but did not affect Ca^<2+>mobilization from internal stores. Fluid flow stimulation (shear stress= 5 dynes/cm^2) increased [Ca^<2+>]_i and enhanced MLC phosphorylation. ML-9 also inhibited Ca^<2+>response and MLC phosphorylation in fluid-flow-stimulated cells. The Ca^<2+>influx in response to BK was linearly correlated with the diphosphorylation of MLC in ML-9 treated cells.
Effects of ML-5 and ML-7, analogues of ML-9, to inhibit Ca^<2+>influx paralleled their potencies to inhibit MLCK activity. A structurally different MLCK infibitor, wortmannin, mimicked the effect of ML-9 on the thapsigargin-stimulated Ca^<2+>response. On the other hand, neither PKC inhibitor nor PKA inhibitor affected the BK-stimulated Ca^<2+>response. These findings demonstrate that MLCK plays an essential role to regulate the plasmalemmal Ca^<2+>influx in agonist-and fluid-flow-stimulated endothelial cells. This study is the first to report the close relationship between the Ca^<2+>influx and MLC diphosphorylation.
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