Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS

The shear-induced intracellular signal transduction pathway in vascular end othelial cells involves tyrosine phosphorylation and activation of mitogen- activated protein (MAP) kinase, which may be responsible for the sustained release of nitric oxide. MAP kinase is known to be activated by reactive ox ygen species (ROS), such as H2O2, in several cell types. ROS production in ligand-stimulated nonphagocytic cells appears to require the participation of a Ras-related small GTP-binding protein, Rac1. We hypothesized that Rac1 might serve as a mediator for the effect of shear stress on MAP kinase act ivation. Exposure of bovine aortic endothelial cells to laminar shear stres s of 20 dyn/cm(2) for 5-30 min stimulated total cellular and cytosolic tyro sine phosphorylation as well as tyrosine phosphorylation of MAP kinase. Tre ating endothelial cells with the antioxidants N-acetylcysteine and pyrrolid ine dithiocarbamate inhibited in a dose-dependent manner the shear-stimulat ed increase in total cytosolic and, specifically, MAP kinase tyrosine phosp horylation. Hence, the onset of shear stress caused an enhanced generation of intracellular ROS, as evidenced by an oxidized protein detection kit, wh ich were required for the shear-induced total cellular and MAP kinase tyros ine phosphorylation. Total cellular and MAP kinase tyrosine phosphorylation was completely blocked in sheared bovine aortic endothelial cells expressi ng a dominant negative Rac1 gene product (N17rac1). We concluded that the G TPase Rac1 mediates the shear-induced tyrosine phosphorylation of MAP kinas e via regulation elf the flow-dependent redox changes in endothelial cells in physiological and pathological circumstances.