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

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Abstract
Objective: Oxidized low-density lipoprotein (ox-LDL) increases superoxide anion (O2−) 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, eNOS Thr495 was constitutively phosphorylated. ox-LDL, but not native LDL, enhanced the production of O2− 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 Thr495. Protein kinase C (PKC) has been reported to phosphorylate this residue, and the increase in the phosphorylation of Thr495 induced by phorbol 12-myristate 13-acetate was attenuated in cells pretreated with ox-LDL. Moreover, the phosphorylation and activity of PKCα was attenuated by ox-LDL and paralleled the changes in eNOS phosphorylation. ox-LDL also induced the dissociation of eNOS from the plasma and Golgi membranes. In COS-7 cells, a T495A eNOS mutant generated significantly more O2− than a T495D mutant did, indicating that the dephosphorylation of Thr495 alone can increase O2− production by eNOS. However, although the dephosphorylation of Thr495 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 PKCα 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 O2-.

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