Nitric oxide activates p21^ras and leads to the inhibition of endothelial NO synthase by protein nitration

Recent data has indicated that exogenous nitric oxide (NO) has the ability to decrease endogenous NO production by inhibiting the enzyme responsible for its generation, NO synthase (NOS). Our previous studies have indicated that increased generation of reactive oxygen species (ROS) play an important role in the inhibitory event. However, the mechanisms for these effects remain unclear. Previous studies have suggested that NO can activate p21^ras. Thus, the objective of this study was to determine whether NO-mediated activation of p21^ras is involved in the inhibitory process, and to further elucidate the involvement of ROS. Using primary cultures of ovine pulmonary arterial endothelial cells we demonstrated that the NO donor SpermineNONOate, increased p21^ras activity by 2.3-fold compared to untreated cells, and that the farnesyl-transferase inhibitor, α-hydroxyfarnesylphosphonic acid, reduced p21^ras activity and significantly reduced inhibition of eNOS. The overexpression of p21^ras increased, while the overexpression of an NO unresponsive mutant of p21^ras (p21^ras C118S) reduced, the inhibition of eNOS by NO. Further, we identified an increase in the level of superoxide and peroxynitrite in endothelial cells exposed to NO that was reduced by p21^ras C118S transient transfection. Conversely, levels of superoxide and peroxynitrite could be increased by the over expression of wild type p21^ras. Similarly, eNOS nitration induced by NO exposure was reduced by p21^ras C118S transient transfection, and increased by the overexpression of wild-type p21^ras. Finally, results also demonstrated that eNOS itself was a significant producer of superoxide, and that this appeared to be related to a p21^ras-dependent increase in phosphorylation of Ser1177. Our results implicate a signaling pathway involving p21^ras activation, superoxide generation, and peroxynitrite formation as being important in the NO-mediated inhibition of eNOS.