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Cited by 381Nitric Oxide and Endothelin EffectsRedox biology and oxidative stressProtein Kinase Regulation and GTPase Signaling

Hydrogen Peroxide Activates Endothelial Nitric-oxide Synthase through Coordinated Phosphorylation and Dephosphorylation via a Phosphoinositide 3-Kinase-dependent Signaling Pathway

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Thomas, Shane R., Chen, Kai, Keaney, John F.·2002·Journal of Biological Chemistry
DOI 10.1074/jbc.m109107200
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Abstract

Endothelial nitric-oxide synthase (eNOS) is an important component of vascular homeostasis. During vascular disease, endothelial cells are exposed to excess reactive oxygen species that can alter cellular phenotype by inducing various signaling pathways. In the current study, we examined the implications of H(2)O(2)-induced signaling for eNOS phosphorylation status and activity in porcine aortic endothelial cells. We found that H(2)O(2) treatment enhanced eNOS activity and NO bioactivity as determined by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline and cellular cGMP content. Concomitant with eNOS activation, H(2)O(2) also activated Akt, increased eNOS phosphorylation at Ser-1177, and decreased eNOS phosphorylation at Thr-495. H(2)O(2)-induced promotion of eNOS activity and modulation of the eNOS phosphorylation status at Ser-1177 and Thr-495 were significantly attenuated by selective inhibitors of Src kinase, the ErbB receptor family, and phosphoinositide 3-kinase (PI 3-K). We found that Akt activation, eNOS Ser-1177 phosphorylation, and eNOS activation by H(2)O(2) were calcium-dependent, whereas eNOS dephosphorylation at Thr-495 was not, suggesting a branch point in the signaling cascade downstream from PI 3-K. Consistent with this, overexpression of a dominant negative isoform of Akt inhibited H(2)O(2)-induced phosphorylation of eNOS at Ser-1177 but not dephosphorylation of eNOS at Thr-495. Together, these data indicate that H(2)O(2) promotes calcium-dependent eNOS activity through a coordinated change in the phosphorylation status of the enzyme mediated by Src- and ErbB receptor-dependent PI 3-K activation. In turn, PI 3-K mediates eNOS Ser-1177 phosphorylation via a calcium- and Akt-dependent pathway, whereas eNOS Thr-495 dephosphorylation does not involve calcium or Akt. This response may represent an attempt by endothelial cells to maintain NO bioactivity under conditions of enhanced oxidative stress.

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  • Intermediate NO levels (50-300 nM) can activate a range of positive and negative responses from wound healing to oncogenic pathways.

    “An emerging paradigm proposes that intermediate levels (50–300 nM) can activate a range of positive and negative responses from wound healing to oncogenic pathways”

    From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [938]

  • Higher NO concentrations (>1 μM) can lead to oxidative stress and nitrative and nitrosative stress via peroxynitrite and nitrosating species, and in combination with oxygen can trigger posttranslational modification of proteins, lipids, and DNA.

    “Higher concentrations of NO (>1 μM) can lead not only to oxidative stress but also nitrative and nitrosative stress via the generation of peroxynitrite and nitrosating species”

    From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [938]

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  1. [2]2. Rees, D. D., Palmer, R. M., and Moncada, S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3375 -3378
  2. [10]10. Michell, B. J., Griffiths, J. E., Mitchelhill, K. I., Rodriguez-Crespo, I., Tiganis, T., Bozinovski, S., de Montellano, P. R., Kemp, B. E., and Pearson, R. B. (1999) Curr. Biol. 9, 845 -848
  3. [24]24. Moro, M. A., Russell, R. J., Cellek, S., Lizasoain, I., Su, Y., Darley-Usmar, V. M., Radomski, M. W., and Moncada, S. (1995) Proc. Natl. Acad. Sci. U. S. A. 93, 1480 -1485
  4. [48]48. Paxinou, E., Weisse, M., Chen, Q., Souza, J. M., Hertkorn, C., Selak, M., Daikhin, E., Yudkoff, M., Sowa, G., Sessa, W. C., and Ischiropoulos, H. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 11575 -11580

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Authors
Thomas, S. R., Chen, K., & Keaney, J. F.
Affiliations
Boston University (US)
Corresponding author
Keaney, John F.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2002-02
Volume
277
Issue
8
Pages
6017-6024
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m109107200
OpenAlex
W2119043221
Cited by
381
References
47
Citations by year
2026: 2 · 2025: 8 · 2024: 4 · 2023: 5 · 2022: 10 · 2021: 12 · 2020: 7 · 2019: 8 · 2018: 9 · 2017: 14 · 2016: 13 · 2015: 20 · 2014: 21 · 2013: 17 · 2012: 19
FWCI
16.21
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (5r01dk055656-03)
  • National Heart, Lung, and Blood Institute (P01HL060886, HL-60886)
  • National Institute of Diabetes and Digestive and Kidney Diseases (DK-55656)
Topics
  • Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
Keywords
hydrogen peroxide, endothelial nitric oxide synthase, eNOS phosphorylation, phosphatidylinositol 3-kinase, Akt signaling, Src kinase, ErbB receptors, calcium signaling, nitric oxide bioactivity, cyclic GMP, reactive oxygen species
MeSH terms
Animals, Aorta, Arginine, Cells, Cultured, Chromones, Citrulline, Egtazic Acid, Endothelium, Vascular, Enzyme Activation, Enzyme Inhibitors, Hydrogen Peroxide, Kinetics, Morpholines, Nitric Oxide, Phosphorylation, Phosphoserine, Phosphothreonine, Quinazolines, Swine, Signal Transduction, Nitric Oxide Synthase, Phosphatidylinositol 3-Kinases, Calcium Signaling, Tyrphostins, Nitric Oxide Synthase Type III

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