Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress.
Endothelial cells release nitric oxide (NO) more potently in response to increased shear stress than to agonists which elevate intracellular free calcium concentration ([Ca2+]i). To determine mechanistic differences in the regulation of endothelial constitutive NO synthase (ecNOS), we measured NO production by bovine aortic endothelial cells exposed to shear stress in a laminar flow chamber or treated with Ca2+ ionophores in static culture. The kinetics of cumulative NO production varied strikingly: shear stress (25 dyne/cm2) stimulated a biphasic increase over control that was 13-fold at 60 minutes, whereas raising [Ca2+]i caused a monophasic 6-fold increase. We hypothesized that activation of a protein kinase cascade mediates the early phase of flow-dependent NO production. Immunoprecipitation of ecNOS showed a 210% increase in phosphorylation 1 minute after flow initiation, whereas there was no significant increase after Ca2+ ionophore treatment. Although ecNOS was not tyrosine-phosphorylated, the early phase of flow-dependent NO production was blocked by genistein, an inhibitor of tyrosine kinases. To determine the Ca2+ requirement for flow-dependent NO production, we measured [Ca2+]i with a novel flow-step protocol. [Ca2+]i increased with the onset of shear stress, but not after a step increase. However, the step increase in shear stress was associated with a potent biphasic increase in NO production rate and ecNOS phosphorylation. These studies demonstrate that shear stress can increase NO production in the absence of increased [Ca2+]i, and they suggest that phosphorylation of ecNOS may importantly modulate its activity during the imposition of increased shear stress.
Corson
M A
MA
Cardiology Division, University of Washington School of Medicine, Seattle 98195, USA. mcorson@u.washington.edu
James
N L
NL
Latta
S E
SE
Nerem
R M
RM
Berk
B C
BC
Harrison
D G
DG
D000818:Animals; D002118:Calcium / Q000819:agonists / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein; D007425:Intracellular Membranes / Q000378:metabolism; D007529:Isoflavones / Q000494:pharmacology; D007700:Kinetics; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D009994:Osmolar Concentration; D010766:Phosphorylation; D013314:Stress, Mechanical
D004791:Enzyme Inhibitors; D007529:Isoflavones; D009569:Nitric Oxide; D019833:Genistein; D019001:Nitric Oxide Synthase; D002118:Calcium