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J. Biol. Chem., Vol. 277, Issue 1, 725-734, January 4, 2002
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From the ¶ Department of Physiology and Biomedical Imaging
Group, University of Massachusetts Medical School,
Worcester, Massachusetts 01655, § Medical and
Biological Laboratories, Ina, Nagano 396, Japan and the Agonist-induced translocation of RhoA and the
spatio-temporal change in myosin regulatory light chain
(MLC20) phosphorylation in smooth muscle was
clarified at the single cell level. We expressed green fluorescent
protein-tagged RhoA in the differentiated tracheal smooth muscle cells
and visualized the translocation of RhoA in a living cell with
three-dimensional digital imaging analysis. The stimulation of the
cells by carbachol initiated the translocation of green fluorescent
protein-tagged wild type RhoA to the plasma membrane within a minute.
The change in MLC20 phosphorylation level after carbachol
stimulation was monitored by using phospho-Ser-19-specific antibody
recognizing the phosphorylated MLC20 in single cells. Cells
expressing the dominant negative form (T19N) of RhoA significantly suppressed sustained MLC20 phosphorylation during the
prolonged phase (>300 s), whereas the maximum phosphorylation level
(reached at 10 s after stimulation) of these cells was not
significantly different from the control cells. The kinetics of RhoA
translocation was consistent with that of sustained myosin
phosphorylation, suggesting the involvement of a RhoA pathway.
Carbachol stimulation increased myosin phosphorylation within a minute
both at the cortical and the central region. On the other hand, during
prolonged phase, myosin phosphorylation was sustained at the cortical
region of the cells but not at the central fibers. A myosin light chain kinase-specific inhibitor, ML-9, diminished myosin phosphorylation at
the central region of the cells after the stimulation but not at the
cortical area. On the other hand, Y-27632, a Rho kinase-specific inhibitor, diminished myosin phosphorylation at the cortical region but
not the central region. The results clearly show that the myosin light
chain kinase pathway and the Rho pathway distinctly change myosin
phosphorylation in smooth muscle cells in both a temporal and spatial manner.
Rho-dependent Agonist-induced Spatio-temporal Change
in Myosin Phosphorylation in Smooth Muscle Cells*
,,
, First
Department of Internal Medicine, University of Tokyo, Hongo, Bunkyo-ku,
Tokyo 113, Japan
*
This work was supported in part by National Institutes of
Health Grants HL60831 and HL61426.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of an American Heart Association postdoctoral fellowship
(New England Affiliate).
Present address: Department of Thoracic Surgery, Mie
University, School of Medicine, Tsu, Mie 514, Japan
**
To whom correspondence should be addressed: Dept. of Physiology,
University of Massachusetts Medical School, 55 Lake Ave. N., Worcester,
MA 01655. Tel.: 508-856-1954; Fax: 508-856-4600; E-mail:
Mitsuo.Ikebe@umassmed.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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