Abstract 221: Phosphorylation and L-Arginine Increase Superoxide Generation from Endothelial Nitric Oxide Synthase (eNOS)
In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) via a calcium/calmodulin dependent reaction. With oxidative or other depletion of the critical cofactor BH4, NOS generates superoxide (•O2−). Phosphorylation of serine 1177 (S1177) in human eNOS has been shown to modulate eNOS derived-NO formation, and recently, we have shown that L-Arg can increase •O2− generation from BH4-free eNOS. However, it is unclear how S1177 phosphorylation regulates •O2− generation from eNOS and if L-Arg functions in a cooperative manner with phosphorylation. In this work, we show that in the presence of excess Ca2+, phosphorylation of S1177 by Akt led to an increase (>50%) in maximal •O2− generation from eNOS. Moreover, this phosphorylation greatly altered the Ca2+ sensitivity decreasing the calcium EC50 of •O2− generation from 22.1 μM to 4.5 μM, such that eNOS-dependent •O2− generation became essentially Ca2+-independent. Additionally, Akt phosphorylated eNOS displayed higher resistance to EGTA inactivation, and a slightly lower EC50 for calmodulin (149 vs 208 nM). In the presence of L-Arg (1 mM), the O2•− production rate from heNOS was increased by 150% compared to that without L-Arg, but when S1177 was phosphorylated L-Arg induced a further increase in •O2− generation of >600%. Using confocal microscopy we show that both S1177 phosphorylation and the addition of L-Arg cooperatively increased •O2− generation from uncoupled eNOS in endothelial cells (Figure⇓). Thus, phosphorylation of eNOS at S1177 and L-Arg are pivotal in the regulation of •O2− and NO generation, altering both the Ca2+ sensitivity of the enzyme and rate of product formation.
This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia)