Abstract 20702: Reoxygenation — Induced Activation of Golgi Resident eNOS Regulates Exocytosis in the Vascular Endothelium: The Role of Cytosolic Phosphatases
Nitric Oxide (NO) elicits biological response by binding to heme moieties. The classic example is the activation of guanylate cyclase leading to smooth muscle relaxation. However, increasing data indicates that NO regulates various proteins and cellular processes such as exocytosis by formation of nitrosothiols. Formation of NO in the endothelium is regulated by post-translational modification of endothelial NO synthase (eNOS). Subcellular localization of eNOS, either Golgi or plasma membrane resident, may also influence the mechanisms of stimulation, the amount of NO produced and its signaling function. BAECs were subjected to anoxia (2 hrs) and reoxygenation (A/R, 30 – 60 mins). We have shown that in response to reoxygenation, as occurs in the reperfused heart, eNOS in endothelial cells is activated due to selective serine 1179 phosphorylation in the Golgi. When the cells are pre-treated with phosphatase inhibitors, PPM1D or Okadaic acid, this selectivity is lost with generalized eNOS phosphorylation seen throughout the cell suggesting a role of cytosolic phosphatases accounting for the targeted Golgi specific eNOS activation. This post-translational modification of Golgi resident eNOS leads to NO production that induces S-nitrosylation of cellular proteins and modulates intracellular protein trafficking and exocytosis of Weibel-Palade bodies (WPB) as detected by the inhibition of von Willibrand Factor (vWF) release and the translocation of P-selectin to the plasma membrane. Conversely, this exocytosis is not inhibited by activation of plasma membrane eNOS (Fig). Agonists known to activate plasma membrane resident eNOS (VEGF 50 nM and acetylcholine 5 μM) did not inhibit the release of vWF. Thus, a discrete intracellular NOS pool is activated by hypoxia and reoxygenation that serves to inhibit exocytosis of WPB in turn decreasing inflammation and thrombosis.
- Oxidative stress
- Endothelium-derived relaxing factor
- Nitric oxide synthase
- Ischemia reperfusion
- Vascular development
- © 2010 by American Heart Association, Inc.