Phosphorylation of Endothelial Nitric Oxide Synthase in Response to Fluid Shear Stress

Abstract

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.

Mechanisms of H2O2-induced oxidative stress in endothelial cells

“…Shear stress has also been shown to increase pro-oxidants, including NOS formation of NO and SOD (Malek et al 1995;Uematsu et al 1995;Corson et al 1996;Dimmeler et al 1999;Woodman et al 1999 under H 2 O 2 -induced oxidative stress under both shear and static conditions. The overall goal of these studies is to provide a possible model for disease development within the vasculature.…”

Nitric oxide generation mediated by ?-adrenoceptors is impaired in platelets from patients with Type 2 diabetes mellitus

“…Alterations in Ca 2+ signalling have been described in diabetes [36,37], although this is unlikely to affect βAR-mediated NOS3 activation, as this activation has been shown to occur without detectable changes in intracellular Ca 2+ either in human vascular endothelial cells [38] or in platelets [6]. It has been reported that sheer stress can activate NOS3 in a Ca 2+ -independent manner [39]. This is due to phosphorylation of the NOS3 serine residue Ser 1177 , which alters the enzyme's sensitivity to Ca 2+ , enabling maximal activity at sub-physiological concentrations of Ca 2+ [40].…”

Dose dependent effects of reactive oxygen and nitrogen species on the function of neuronal nitric oxide synthase

“…These include protein-protein interactions [22][23][24][25][26], protein phosphorylation [27][28][29], endogenous inhibitory N-methylated L-arginines [30,31], subcellular localization [32], subunit dimerization [33], as well as product feedback inhibition, by which excess NO down-regulates the amount of subsequent NO synthesis. NOS activity can also be altered by oxidative stress.…”