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

Cardiovascular Diabetology

Original Investigation

Lingyun Wu¹  Lixin Liu²  Syed M Qadri²  Yang Su² 

[1] Department of Health Sciences, Lakehead University and Thunder Bay Regional Research Institute, Thunder Bay, ON, Canada;Department of Pharmacology, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, Canada;

关键词: Methylglyoxal;    eNOS uncoupling;    Superoxide;    Tyrosine nitration;    Biopterins;    eNOS phosphorylation;   

DOI  :  10.1186/1475-2840-12-134

 received in 2013-07-25, accepted in 2013-09-02,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundIncreased 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 superoxideO2•- production, phosphorylation status and eNOS uncoupling in EA.hy926 human endothelial cells.MethodsIn 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.O2•-–dependent fluorescence was determined using a commercially available kit and tissue biopterin levels were measured by fluorometric HPLC analysis.ResultsIn EA.hy926 cells, MG treatment significantly enhancedO2•- 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-derivedO2•- generation but did not modify eNOS phosphorylation or monomerization.ConclusionMG triggers eNOS uncoupling and hypophosphorylation in EA.hy926 endothelial cells associated withO2•- generation and biopterin depletion. The observed effects of the glycolysis metabolite MG presumably account, at least in part, for endothelial dysfunction in diabetes.

【 授权许可】

Unknown   
© Su et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.