Oscillatory shear stress upregulation of endothelial nitric oxide synthase requires intracellular hydrogen peroxide and CaMKII.

We have previously shown that hydrogen peroxide (H(2)O(2)) upregulates endothelial nitric oxide synthase (eNOS) expression via a calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated mechanism whereas it also acutely activates eNOS enzyme. We hypothesized that oscillatory shear stress (OSS), which stimulates endogenous H(2)O(2), would have effects on eNOS expression and function similar to that of exogenous H(2)O(2). Exposure of bovine aortic endothelial cells to OSS (+/-15 dynes/cm(2)) increased eNOS mRNA expression by 3-fold. Pretreatment with either polyethylene glycol-catalase (PEG-CAT, a scavenger of H(2)O(2)) or KN93, an inhibitor of CaMKII, abolished this response. OSS activated CaMKII in an H(2)O(2)-dependent fashion whereas unidirectional laminar shear stress (LSS) inhibited CaMKII phosphorylation. Inhibition of c-Src (essential for LSS upregulation of eNOS) had no effect on OSS upregulation of eNOS. Additionally, OSS stimulated NO* production acutely. Scavenging of H(2)O(2) by PEG-CAT attenuated OSS stimulation of NO* by 50% whereas it had no effect on LSS regulation of NO* production. These data suggest that intracellular H(2)O(2) and CaMKII mediate OSS upregulation of eNOS. The acute activation of eNOS by OSS also partially requires H(2)O(2). As OSS has been shown previously to stimulate sustained production of superoxide (O(2)*-) which would inactivate NO*, these responses may represent attempted compensation to restore NO* bioavailability in areas exposed to OSS. Simultaneous stimulation of O(2)*- and NO* by this mechanism, however, could facilitate peroxynitrite formation and protein nitration, which may enhance atherosclerotic lesion formation. Both OSS and LSS upregulate eNOS expression but via different signaling mechanisms.