Endothelial Nitric Oxide Synthase Phosphorylation at Threonine 495 and Mitochondrial Reactive Oxygen Species Formation in Response to a High H2O2 Concentration

TY - JOUR

T1 - Endothelial Nitric Oxide Synthase Phosphorylation at Threonine 495 and Mitochondrial Reactive Oxygen Species Formation in Response to a High H2O2 Concentration

AU - Guterbaum, Thomas Jeremy

AU - Braunstein, Thomas Hartig

AU - Fossum, A

AU - von Holstein-Rathlou, Niels-Henrik

AU - Torp-Pedersen, C T

AU - Dominguez Vall-Lamora, Maria Helena

PY - 2013/10

Y1 - 2013/10

N2 - Background: Hydrogen peroxide (H2O2) is produced in vessels during ischemia/reperfusion and during inflammation, both leading to vascular dysfunction. We investigated cellular pathways involved in endothelial nitric oxide synthase (eNOS) phosphorylation at Threonine 495 (Thr 495) in human umbilical vein endothelial cells (HUVECs) exposed to H2O2. Methods: HUVECs were exposed to 400 μM H 2O2 for 30 min. Phosphorylation at Thr495 was assessed by Western blotting and reactive oxygen species (ROS) monitored by flow cytometry. Protein kinase C (PKC) pathways were investigated by pretreatment with PKC-β inhibitor ruboxistaurin or pan-PKC inhibitor GF109203X. In addition, we investigated ROCK and ERK pathways by MEKK1/2 inhibitor U0126 and ROCK inhibitor Y27632. Results: H2O2 increased eNOS phosphorylation at Thr495 (to 176% vs. control (100%), p < 0.001) along with increased mitochondrial ROS formation (from 19.7 to 45.3%, p < 0.01). This rise in phosphorylation could be prevented by U0126 and Y27632 in a dose-dependent manner, but did not result in lowered mitochondrial ROS formation. Conversely, addition of the antioxidant N-acetyl-L-cysteine only prevented mitochondrial ROS formation but did not prevent phosphorylation of eNOS Thr495. Conclusion: H2O2-mediated phosphorylation of eNOS Thr495 is mediated by ROCK and ERK activity, but not by PKC, and is uncoupled from mitochondrial ROS signaling. Furthermore, ERK inhibition increased mitochondrial ROS formation.

AB - Background: Hydrogen peroxide (H2O2) is produced in vessels during ischemia/reperfusion and during inflammation, both leading to vascular dysfunction. We investigated cellular pathways involved in endothelial nitric oxide synthase (eNOS) phosphorylation at Threonine 495 (Thr 495) in human umbilical vein endothelial cells (HUVECs) exposed to H2O2. Methods: HUVECs were exposed to 400 μM H 2O2 for 30 min. Phosphorylation at Thr495 was assessed by Western blotting and reactive oxygen species (ROS) monitored by flow cytometry. Protein kinase C (PKC) pathways were investigated by pretreatment with PKC-β inhibitor ruboxistaurin or pan-PKC inhibitor GF109203X. In addition, we investigated ROCK and ERK pathways by MEKK1/2 inhibitor U0126 and ROCK inhibitor Y27632. Results: H2O2 increased eNOS phosphorylation at Thr495 (to 176% vs. control (100%), p < 0.001) along with increased mitochondrial ROS formation (from 19.7 to 45.3%, p < 0.01). This rise in phosphorylation could be prevented by U0126 and Y27632 in a dose-dependent manner, but did not result in lowered mitochondrial ROS formation. Conversely, addition of the antioxidant N-acetyl-L-cysteine only prevented mitochondrial ROS formation but did not prevent phosphorylation of eNOS Thr495. Conclusion: H2O2-mediated phosphorylation of eNOS Thr495 is mediated by ROCK and ERK activity, but not by PKC, and is uncoupled from mitochondrial ROS signaling. Furthermore, ERK inhibition increased mitochondrial ROS formation.

U2 - 10.1159/000354225

DO - 10.1159/000354225

M3 - Journal article

C2 - 24008236

SN - 1018-1172

VL - 50

SP - 410

EP - 420

JO - Journal of Vascular Research

JF - Journal of Vascular Research

IS - 5

ER -