Critical role of hydrogen peroxide signaling in the sequential activation of p38 MAPK and eNOS in laminar shear stress.

Laminar shear stress (LSS) is a protective hemodynamic regulator of endothelial function and limits the development of atherosclerosis and other vascular wall diseases related to pathophysiological generation of reactive oxygen species. LSS activates several endothelial signaling responses, including the activation of MAPKs and eNOS. Here, we explored the mechanisms of activation of these key endothelial signaling pathways. Using the cone/plate model we found that LSS (12 dyn/cm(2)) rapidly promotes endothelial intracellular generation of superoxide and hydrogen peroxide (H(2)O(2)). Physiological concentrations of H(2)O(2) (flux of 0.1 nM/min and 15 µM added extracellularly) significantly activated both eNOS and p38 MAPK. Pharmacological inhibition of NADPH oxidases (NOXs) and specific knockdown of NOX4 decreased LSS-induced p38 MAPK activation. Whereas the absence of eNOS did not alter LSS-induced p38 MAPK activation, pharmacological inhibition and knockdown of p38α MAPK blocked H(2)O(2)- and LSS-induced eNOS phosphorylation and reduced (•)NO levels. We propose a model in which LSS promotes the formation of signaling levels of H(2)O(2), which in turn activate p38α MAPK and then stimulate eNOS, leading to increased (•)NO generation and protection of endothelial function.

S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities.

Nitric oxide is implicated in a variety of signaling pathways in different systems, notably in endothelial cells. Some of its effects can be exerted through covalent modifications of proteins and, among these modifications, increasing attention is being paid to S-nitrosylation as a signaling mechanism. In this work, we show by a variety of methods (ozone chemiluminescence, biotin switch, and mass spectrometry) that the molecular chaperone Hsp90 is a target of S-nitrosylation and identify a susceptible cysteine residue in the region of the C-terminal domain that interacts with endothelial nitric oxide synthase (eNOS). We also show that the modification occurs in endothelial cells when they are treated with S-nitroso-l-cysteine and when they are exposed to eNOS activators. Hsp90 ATPase activity and its positive effect on eNOS activity are both inhibited by S-nitrosylation. Together, these data suggest that S-nitrosylation may functionally regulate the general activities of Hsp90 and provide a feedback mechanism for limiting eNOS activation.