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

Thomas Jeremy Guterbaum; Thomas Hartig Braunstein; A Fossum; Niels-Henrik von Holstein-Rathlou; C T Torp-Pedersen; Maria Helena Dominguez Vall-Lamora

doi:10.1159/000354225

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

Thomas Jeremy Guterbaum, Thomas Hartig Braunstein, A Fossum, Niels-Henrik von Holstein-Rathlou, C T Torp-Pedersen, Maria Helena Dominguez Vall-Lamora

15 Citations (Scopus)

Abstract

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.

Original language	English
Journal	Journal of Vascular Research
Volume	50
Issue number	5
Pages (from-to)	410-420
Number of pages	11
ISSN	1018-1172
DOIs	https://doi.org/10.1159/000354225
Publication status	Published - Oct 2013

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Guterbaum, T. J., Braunstein, T. H., Fossum, A., von Holstein-Rathlou, N.-H., Torp-Pedersen, C. T., & Dominguez Vall-Lamora, M. H. (2013). Endothelial Nitric Oxide Synthase Phosphorylation at Threonine 495 and Mitochondrial Reactive Oxygen Species Formation in Response to a High H2O2 Concentration. Journal of Vascular Research, 50(5), 410-420. https://doi.org/10.1159/000354225

Endothelial Nitric Oxide Synthase Phosphorylation at Threonine 495 and Mitochondrial Reactive Oxygen Species Formation in Response to a High H2O2 Concentration. / Guterbaum, Thomas Jeremy; Braunstein, Thomas Hartig; Fossum, A et al.
In: Journal of Vascular Research, Vol. 50, No. 5, 10.2013, p. 410-420.

Research output: Contribution to journal › Journal article › Research › peer-review

Guterbaum, TJ, Braunstein, TH, Fossum, A, von Holstein-Rathlou, N-H , Torp-Pedersen, CT & Dominguez Vall-Lamora, MH 2013, 'Endothelial Nitric Oxide Synthase Phosphorylation at Threonine 495 and Mitochondrial Reactive Oxygen Species Formation in Response to a High H2O2 Concentration', Journal of Vascular Research, vol. 50, no. 5, pp. 410-420. https://doi.org/10.1159/000354225

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

abstract = "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.",

author = "Guterbaum, {Thomas Jeremy} and Braunstein, {Thomas Hartig} and A Fossum and {von Holstein-Rathlou}, Niels-Henrik and Torp-Pedersen, {C T} and {Dominguez Vall-Lamora}, {Maria Helena}",

year = "2013",

month = oct,

doi = "10.1159/000354225",

language = "English",

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pages = "410--420",

journal = "Journal of Vascular Research",

issn = "1018-1172",

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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 -

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

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