Regulation of ca2+-dependent nitric oxide synthase in bovine aortic
endothelial cells.
Buckley, Barbara J., Zermeena Mirza, and A. Richard Whorton.
Departments of Pharmacology and Medicine, Duke University Medical
Center, Durham, NC 27710
APStracts 2:0157C, 1995.
Vascular endothelium responds to Ca2+-mobilizing agonists by producing
NO, a potent vasodilator and inhibitor of platelet aggregation.
Regulation of eNOS in intact cells is not well understood. We
investigated the kinetics of NO formation in response to Ca2+
-mobilizing agonists, the requirement for extracellular L-arginine and
the role of NO in regulating eNOS activity. When endothelial cells
were stimulated with bradykinin and ATP in the presence of 100 NM L
-arginine, we observed a rapid and transient rise in intracellular
Ca2+ ([Ca2+]i) from 50 +/- 8 nM to 698 +/- 74 nM and 637 +/- 53 nM,
respectively, and a rapid and transient rise in NO production from a
basal level of 37 pmol/min/mg protein to 256 and 275 pmol/min/mg
protein, respectively. When cells were stimulated with A23187 or
thapsigargin (TG) in the presence of 100 NM L-arginine, we observed a
sustained increase in [Ca2+]i and a sustained increase in NO
production. The rate of NO synthesis was linear over 30 min, rising
above control levels of 7 pmol/min to 53 pmol/min for A23187 and 62
pmol/min for TG. TG stimulated NO production and [Ca2+]i with EC50
values of 0.01 and 0.05 NM, respectively. Ca2+-stimulated NO
production was attenuated by the NOS inhibitor LNMMA, the removal of
extracellular L-arginine and the Ca2+-chelator EGTA. When we exposed
cells to NO gas (3.1 mM for 15 min) and S-nitrosoglutathione (10 mM
for 15 min), TG-stimulated NO production was decreased by 50%. These
studies in intact cells demonstrate that eNOS activity is tightly
coupled to Ca2+, mirroring agonist-stimulated rises and falls in
[Ca2+]i. In addition, eNOS requires supplementation with low levels
of extracellular L-arginine to support a full response. Finally,
while eNOS is inhibited by high doses of exogenous NO, it is not
sensitive to feedback inhibition by endogenous NO.
Received 23 January 1995; accepted in final form 21 March 1995.
APS Manuscript Number C43-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 10 April 1995.