Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low-density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells.

BACKGROUND AND PURPOSE

One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O(2) (•-) ) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism.

EXPERIMENTAL APPROACH

HUVECs were incubated with ox-LDL with/without pyridoxine, N(G) -nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O(2) (•-) was measured using lucigenin chemiluminescence, and O(2) (•-) -sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCα and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCα/βII, total PKC, phospho-PKCα, total PKCα and p47phox were measured by Western blot.

KEY RESULTS

Ox-LDL significantly increased O(2) (•-) production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O(2) (•-) generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCα. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments.

CONCLUSIONS AND IMPLICATIONS

Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCα, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction.

© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.