High Glucose Attenuates Protein S-Nitrosylation in Endothelial Cells

Role of Oxidative Stress

  1. Carol Wadham1,
  2. Angela Parker1,
  3. Lijun Wang2 and
  4. Pu Xia1,2
  1. 1Signal Transduction Laboratory, Division of Human Immunology, Hanson Institute, Institute of Medical and Veterinary Science, Adelaide, Australia;
  2. 2Signal Transduction Laboratory, Centenary Institute, and Faculty of Medicine, the University of Sydney, Sydney, Australia.
  1. Address correspondence and reprint requests to Pu Xia, MD, Signal Transduction Laboratory, Centenary Institute, Locked Bag 6, Newtown, NSW 2042, Australia. E-mail: p.xia{at}centenary.org.au.

Abstract

OBJECTIVE Hyperglycemia-induced endothelial dysfunction, via a defect of nitric oxide (NO) bioactivity and overproduction of superoxide, is regarded as one of the most significant events contributing to the vascular lesions associated with diabetes. However, the mechanisms underlying such hyperglycemic injury remain undefined. We hypothesized that alterations in cellular protein S-nitrosylation may contribute to hyperglycemia-induced endothelial dysfunction.

RESEARCH DESIGN AND METHODS We exposed endothelial cells to high glucose in the presence and absence of reactive oxygen species inhibitors and used the biotin switch assay to analyze the alteration in the global pattern of protein S-nitrosylation compared with cells cultured under normal glucose conditions. We identified endogenous S-nitrosylated proteins by mass spectrometry and/or immunoblotting with specific antibodies.

RESULTS High-glucose treatment induced a significant reduction of endogenous S-nitrosylated proteins that include endothelial NO synthase, β-actin, vinculin, diacylglycerol kinase-α, GRP78, extracellular signal–regulated kinase 1, and transcription factor nuclear factor-κB (NF-κB). Interestingly, these changes were completely reversed by inhibition of superoxide production, suggesting a key role for oxidative stress in the regulation of S-nitrosylation under hyperglycemic conditions. In addition, we found that in parallel with the restoration of decreased S-nitrosylation of NF-κB, high glucose–induced NF-κB activation was blocked by the superoxide inhibitors.

CONCLUSIONS The alterations in protein S-nitrosylation may underlie the adverse effect of hyperglycemia on the vasculature, such as endothelial dysfunction and the development of diabetic vascular complications.

Footnotes

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  • Abbreviations:
    4-OHCA
    α-cyano-4-hydroxycinnamic acid
    CCCP
    carbonyl cyanide m-chlorophenyl hydrazone
    DPI
    diphenyleneiodonium
    eNOS
    endothelial nitric oxide synthase
    FITC
    fluorescein isothiocyanate
    GSNO
    nitrosylated glutathione
    GSH
    reduced glutathione
    HEN
    HEPES, EDTA, neocuproine
    HUVEC
    human umbilical vein endothelial cell
    IKKβ
    inhibitory κB kinase-β
    mETC
    mitochondrial electron transport chain
    NF-κB
    nuclear factor-κB
    NOS
    nitric oxide synthase
    NOX
    NAD(P)H oxidase
    oxLDL
    oxidized LDL
    ROS
    reactive oxygen species
    TNF-α
    tumor necrosis factor-α
    TTFA
    thenoyltrifluoroacetone.
    • Received September 13, 2006.
    • Accepted August 12, 2007.
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This Article

  1. Diabetes vol. 56 no. 11 2715-2721
  1. All Versions of this Article:
    1. db06-1294v1
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