Abstract 225: Intracellular Protein Trafficking In The Vascular Endothelium Is Regulated By Golgi Resident Nitric Oxide Sythase
Endothelial cell-derived nitric oxide (NO) is a critical determinant of cardiovascular homeostasis. Post-translational modification of NO synthases (NOS) effectively modulates enzyme activity, tightly controlling cellular function by production of NO at the right time, location and amount. Recent studies provide evidence for a regulatory role of eNOS phosphorylation in modulating NOS activity either independently or in conjunction with its subcellular localization. Here, we show that in response to reoxygenation, as occurs in the reperfused heart, Golgi resident eNOS in endothelial cells is phosphorylated at a critical serine residue that increases eNOS activity as evidenced by confocal microscopy (Figure⇓) and immunoblotting. Further, we find that this phosphorylation is mediated by the PI3K/Akt pathway, and this post-translational modification leads to increased NO production as detected by spin-trapping EPR spectroscopy. Moreover, the observed reoxygenation-dependent increase in NO from the Golgi-resident phospho-eNOS leads to S-nitrosylation of cellular proteins, and modulates intracellular protein trafficking, inhibiting the release of von Willibrand Factor (vWF) and the translocation of P-selectin to the plasma membrane. These findings demonstrate that the PI3K/Akt pathway and subcellular localization of eNOS play critical roles in regulating how NO modulates endothelial cellular function in response to reoxygenation.