Comparison of Protein Expression of the RhoA/ROCK Ca2+-Sensitization Pathway in both Cytosolic and Membrane Fractions of Murine Colon Smooth Muscle
Loading...
Authors
Davidek, Nicole M.
Issue Date
2011
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
The RhoA/ROCK pathway has been targeted for study for the treatment of
smooth muscle disorders, including gastrointestinal (GI) motility disorders. Since [Ca2+]
in smooth muscle tissue initiates contraction, the RhoA/ROCK pathway allows for
continual contraction at any level of Ca2+ within the cell. Using MYPT1, MYPT-pT696,
MYPT1-pT853, LC20, pS19, and Rho kinase (ROCK 2), key components of
RhoA/ROCK regulation of smooth muscle contraction, colon smooth muscle was
analyzed for changes in MYPT1 and LC20 phosphorylation in both the membrane and
cytosolic fractions. Carbachol (CCh), the Rho kinase inhibitor, SR-43677, and the PI3-
kinase inhibitor, LY294002, were utilized to further determine RhoA/ROCK’s effect on
MYPT1 and LC20 phosphorylation. Overall, higher levels of MYPT1, MYPT1-pT696
and pT853, and ROCK2 were found in the membrane fraction of the colon smooth
muscle lysates. LC20 and pS19 were shown to have higher basal levels in the cytosolic
fraction of the colon smooth muscle lysates. CCh caused an overall increase in the levels
of MYPT1, MYPT1-pT696 and pT853, ROCK2, LC20, and pS19 with increasing
exposure to CCh. Treatment with SR-43677 caused a decrease in MYPT1-pT853 in both
fractions of the lysates, with MYPT1 showing a slight decrease and MYPT1-pT696
showing an increase in the pellet. LY294002 treatment did not have any significant effect
on MYPT1, or MYPT1-pT853 or pT696, but overall caused a decrease in expression in
the membrane fraction. Our results indicate that the RhoA/ROCK pathway is membranedependent,
with its downstream effectors translocating to the membrane upon
RhoA/ROCK activation.
Description
Citation
Publisher
License
In Copyright(All Rights Reserved)