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Open Access Original investigation

Methylglyoxal modulates endothelial nitric oxide synthase-associated functions in EA.hy926 endothelial cells

Yang Su1, Syed M Qadri1, Lingyun Wu2 and Lixin Liu1*

Author Affiliations

1 Department of Pharmacology, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, Canada

2 Department of Health Sciences, Lakehead University and Thunder Bay Regional Research Institute, Thunder Bay, ON, Canada

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Cardiovascular Diabetology 2013, 12:134  doi:10.1186/1475-2840-12-134

Published: 19 September 2013

Abstract

Background

Increased levels of the sugar metabolite methylglyoxal (MG) in vivo were shown to participate in the pathophysiology of vascular complications in diabetes. Alterations of endothelial nitric oxide synthase (eNOS) activity by hypophosphorylation of the enzyme and enhanced monomerization are found in the diabetic milieu, and the regulation of this still remains undefined. Using various pharmacological approaches, we elucidate putative mechanisms by which MG modulates eNOS-associated functions of MG-stimulated superoxide <a onClick="popup('http://www.cardiab.com/content/12/1/134/mathml/M1','MathML',630,470);return false;" target="_blank" href="http://www.cardiab.com/content/12/1/134/mathml/M1">View MathML</a> production, phosphorylation status and eNOS uncoupling in EA.hy926 human endothelial cells.

Methods

In cultured EA.hy926 endothelial cells, the effects of MG treatment, tetrahydrobiopterin (BH4; 100 μM) and sepiapterin (20 μM) supplementation, NOS inhibition by NG-nitro-L-arginine methyl ester (L-NAME; 50 μM), and inhibition of peroxynitrite (ONOO-) formation (300 μM Tempol plus 50 μM L-NAME) on eNOS dimer/monomer ratios, Ser-1177 eNOS phosphorylation and 3-nitrotyrosine (3NT) abundance were quantified using immunoblotting. <a onClick="popup('http://www.cardiab.com/content/12/1/134/mathml/M2','MathML',630,470);return false;" target="_blank" href="http://www.cardiab.com/content/12/1/134/mathml/M2">View MathML</a>dependent fluorescence was determined using a commercially available kit and tissue biopterin levels were measured by fluorometric HPLC analysis.

Results

In EA.hy926 cells, MG treatment significantly enhanced <a onClick="popup('http://www.cardiab.com/content/12/1/134/mathml/M3','MathML',630,470);return false;" target="_blank" href="http://www.cardiab.com/content/12/1/134/mathml/M3">View MathML</a> generation and 3NT expression and reduced Ser-1177 eNOS phosphorylation, eNOS dimer/monomer ratio and cellular biopterin levels indicative of eNOS uncoupling. These effects were significantly mitigated by administration of BH4, sepiapterin and suppression of ONOO- formation. L-NAME treatment significantly blunted eNOS-derived <a onClick="popup('http://www.cardiab.com/content/12/1/134/mathml/M4','MathML',630,470);return false;" target="_blank" href="http://www.cardiab.com/content/12/1/134/mathml/M4">View MathML</a> generation but did not modify eNOS phosphorylation or monomerization.

Conclusion

MG triggers eNOS uncoupling and hypophosphorylation in EA.hy926 endothelial cells associated with <a onClick="popup('http://www.cardiab.com/content/12/1/134/mathml/M5','MathML',630,470);return false;" target="_blank" href="http://www.cardiab.com/content/12/1/134/mathml/M5">View MathML</a> generation and biopterin depletion. The observed effects of the glycolysis metabolite MG presumably account, at least in part, for endothelial dysfunction in diabetes.

Keywords:
Methylglyoxal; eNOS uncoupling; Superoxide; Tyrosine nitration; Biopterins; eNOS phosphorylation