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


RESULTS
In unstimulated human endothelial cells, eNOS 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 PKCalpha 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 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 PKCalpha 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)(-).
Fleming
Ingrid
I
Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. fleming@em.uni-frankfurt.de
Mohamed
Annisuddin
A
Galle
Jan
J
Turchanowa
Ljudmila
L
Brandes
Ralf P
RP
Fisslthaler
Beate
B
Busse
Rudi
R
D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000502:physiology; D051571:Protein Kinase C-alpha; D015398:Signal Transduction / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism*; D013552:Swine
D008077:Lipoproteins, LDL; D008244:Lysophosphatidylcholines; C099252:oxidized low density lipoprotein; D013481:Superoxides; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D006152:Cyclic GMP