Abstract 16962: Endoplasmic Reticulum Ca2+ Release Triggers Endothelial NO Synthase Serine 635 Phosphorylation and Activity through ERK1/2
Endothelial NO synthase, eNOS, plays a central role in cardiovascular regulation. The function of eNOS is critically modulated by the phosphorylation of its Ser1179/1177, Ser635/633, and Thr497/495 (bovine/human) residues. In particular, the phosphorylation of eNOS Ser635 by protein kinase A (PKA) has been shown to be an important mechanism underlying the regulation of endothelial function by share stress. Here we show that endoplasmic reticulum (ER) Ca2+ release activates eNOS by selectively promoting its Ser635 phosphorylation. With cultured bovine endothelial cells, discharging Ca2+ from ER by thapsigargin (0.01–10 μM) caused a dose-dependent increase of eNOS Ser635 phosphorylation and increases in NO production. This was a specific action because the phosphorylation status of eNOS Ser1179 or Thr497 was not altered in thapsigargin-treated cells. Mutating the Ser635 residue to a non-phosphorylatable alanine prevented the effect of thapsigargin on eNOS. Blocking ERK1/2 abolished ER Ca2+ release-induced eNOS Ser635 phosphorylation, whereas inhibiting PKA or Ca2+/calmodulin dependent protein kinase II had no effect. Protein phosphorylation assay further confirmed that purified ERK directly phosphorylated recombinant eNOS at its Ser635 residues in vitro. Finally, ER Ca2+ release-induced ERK1/2 activation was demonstrated to mediate the enhancing action of ATP on eNOS Ser635 phosphorylation in endothelial cells. Taken together, these studies reveal that ER Ca2+ release selectively enhances eNOS Ser635 phosphorylation and function via ERK1/2 activation. Since ER Ca2+ is mobilized by various agonists or physicochemical stimuli, the identified ER Ca2+-ERK1/2-eNOS Ser635 phosphorylation pathway may have a broad role in the regulation of endothelial function.
- © 2010 by American Heart Association, Inc.