Regulation of endothelial nitric oxide synthase by the actin cytoskeleton.
Su, Yunchao, Sophia Edwards-Bennett, Michael R. Bubb, and Edward R. Block.
1Department of Medicine, University of Florida College of Medicine; and 2Research
Service, Malcolm Randall Veterans Affairs Medical Center, Gainesville, Florida
32608«hyphen»1197
APStracts 10:0075C, 2003.
In the present study, the association of endothelial nitric oxide synthase (eNOS) with the
actin cytoskeleton in pulmonary artery endothelial cells (PAEC) was examined. We
found that the protein contents of eNOS, actin, and caveolin-1 were significantly higher
in the caveolar fraction of plasma membranes than in the noncaveolar fraction of plasma
membranes in PAEC. Immunoprecipitation of eNOS from lysates of caveolar fractions of
plasma membranes in PAEC resulted in the coprecipitation of actin, and
immunoprecipitation of actin from lysates of caveolar fractions resulted in the
coprecipitation of eNOS. Confocal microscopy of PAEC, in which eNOS was labeled
with fluorescein, F-actin was labeled with Texas red-phalloidin, and G-actin was labeled
with deoxyribonuclease I conjugated with Texas red, also demonstrated an association
between eNOS and F-actin or G-actin. Incubation of purified eNOS with purified F-actin
and G-actin resulted in an increase in eNOS activity. The increase in eNOS activity
caused by G-actin was much higher than that caused by F-actin. Incubation of PAEC
with swinholide A, an actin filament disruptor, resulted in an increase in eNOS activity,
eNOS protein content, and association of eNOS with G-actin and in a decrease in the
association of eNOS with F-actin. The increase in eNOS activity was higher than that in
eNOS protein content in swinholide A-treated cells. In contrast, exposure of PAEC to
phalloidin, an actin filament stabilizer, caused decreases in eNOS activity and association
of eNOS with G-actin and increases in association of eNOS with F-actin. These results
suggest that eNOS is associated with actin in PAEC and that actin and its polymerization
state play an important role in the regulation of eNOS activity.
Received 29 May 2002; accepted in final form 23 January 2003
APS Manuscript Number C248-2.
Article publication pending Am J Physiol Cell Physiol
ISSN 1080-4757 Copyright 2003 The American Physiological Society.
Published in APStracts on 25 March 2003