© 1996 American Heart Association, Inc.
Articles |
Phosphorylation of Endothelial Nitric Oxide Synthase in Response to Fluid Shear Stress
the Cardiology Divisions of the University of Washington School of Medicine (M.A.C., B.C.B.), Seattle, Wa; Emory University School of Medicine (S.E.L., D.G.H.), Atlanta, Ga; and the School of Mechanical Engineering (N.L.J., R.M.N.), Georgia Institute of Technology, Atlanta, Ga.
Correspondence to Marshall A. Corson, MD, Cardiology Division, Box 357710, University of Washington, Seattle, WA 98195. E-mail mcorson@u.washington.edu.
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.
Key Words: mechanotransduction • endothelium-derived relaxing factor • vasorelaxation • Ca2+ • tyrosine kinase
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