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

[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.

[发布日期] 2013-09-19

[关键词] Methylglyoxal;eNOS uncoupling;Superoxide;Tyrosine nitration;Biopterins;eNOS phosphorylation