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 diff erences in the regulation of endothelial constitutive NO synthase (ecN OS), we measured NO production by bovine aortic endothelial cells expo sed to shear stress in a laminar how chamber or treated with Ca2+ iono phores in static culture. The kinetics of cumulative NO production var ied strikingly: shear stress (25 dyne/cm(2)) stimulated a biphasic inc rease over control that was 13-fold at 60 minutes, whereas raising [Ca 2+](i) caused a monophasic 6-fold increase. We hypothesized that activ ation of a protein kinase cascade mediates the early phase of flow-dep endent NO production. Immunoprecipitation of ecNOS showed a 210% incre ase in phosphorylation 1 minute after flow initiation, whereas there w as no significant increase after Ca2+ ionophore treatment. Although ec NOS was not tyrosine-phosphorylated, the early phase of flow-dependent NO production was blocked by genistein, an inhibitor of tyrosine kina ses. To determine the Ca2+ requirement for flow-dependent NO productio n, we measured [Ca2+](i) with a novel flow-step protocol, [Ca2+](i) in creased with the onset of shear stress, but not after a step increase. However, the step increase in shear stress was associated with a pote nt 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 phosphor ylation of ecNOS may importantly modulate its activity during impositi on of increased shear stress.