Oxidized low-density lipoprotein increases superoxide production by endothelial nitric oxide synthase by inhibiting PKCα

Objective: Oxidized low-density lipoprotein (ox-LDL) increases superoxide anion (O-2(-)) production by the endothelial nitric oxide (NO) synthase (eNOS). We assessed whether the uncoupling of eNOS was associated with alterations in eNOS phosphorylation and/or the assembly of the eNOS signaling complex. Methods and results: In unstimulated human endothelial cells, cNOS Thr(495) was constitutively phosphorylated. ox-LDL, but not native LDL, enhanced the production of O-2(-) by endothelial cells, an effect that was partially sensitive to NOS inhibition. ox-LDL, but not native LDL, induced a time- and concentration-dependent decrease in the phosphorylation of eNOS on Thr(495). Protein kinase C (PKC) has been reported to phosphorylate this residue, and the increase in the phosphorylation of Thr(495) induced by phorbol 12-myristate 13-acetate was attenuated in cells pretreated with ox-LDL. Moreover, the phosphorylation and activity of PKC alpha was attenuated by ox-LDL and paralleled the changes in eNOS phosphorylation. ox-LDL also induced the dissociation of cNOS from the plasma and Golgi membranes. In COS-7 cells, a T495A eNOS mutant generated significantly more O-2(-) than a T495D mutant did, indicating that the dephosphorylation of Thr(495) alone can increase O-2(-) production by eNOS. However, although the dephosphorylation of Thr(495) in histamine-stimulated endothelial cells enhanced the binding of calmodulin to eNOS, calmodulin no longer bound to eNOS from ox-LDL-treated endothelial cells. Conclusions: These results indicate that a decrease in the activity of PKC alpha in ox-LDL-treated endothelial cells is associated with the dephosphorylation of eNOS, dissociation of the eNOS signaling complex, and the enhanced production of eNOS-derived O-2(-). (C) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.