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

<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Increased levels of the sugar metabolite methylglyoxal (MG)<jats:italic>in vivo</jats:italic>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<jats:inline-formula><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mfenced><mml:mrow><mml:msubsup><mml:mi>O</mml:mi><mml:mn>2</mml:mn><mml:mrow><mml:mo>•</mml:mo><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:mrow></mml:mfenced></mml:mrow></mml:math></jats:inline-formula>production, phosphorylation status and eNOS uncoupling in EA.hy926 human endothelial cells.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>In cultured EA.hy926 endothelial cells, the effects of MG treatment, tetrahydrobiopterin (BH4; 100 μM) and sepiapterin (20 μM) supplementation, NOS inhibition by N<jats:sup>G</jats:sup>-nitro-L-arginine methyl ester (L-NAME; 50 μM), and inhibition of peroxynitrite (ONOO<jats:sup>-</jats:sup>) 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.<jats:inline-formula><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mrow><mml:msubsup><mml:mi>O</mml:mi><mml:mn>2</mml:mn><mml:mrow><mml:mo>•</mml:mo><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:mrow><mml:mo>–</mml:mo></mml:mrow></mml:math></jats:inline-formula>dependent fluorescence was determined using a commercially available kit and tissue biopterin levels were measured by fluorometric HPLC analysis.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>In EA.hy926 cells, MG treatment significantly enhanced<jats:inline-formula><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mrow><mml:msubsup><mml:mi>O</mml:mi><mml:mn>2</mml:mn><mml:mrow><mml:mo>•</mml:mo><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:mrow></mml:mrow></mml:math></jats:inline-formula>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<jats:sup>-</jats:sup>formation. L-NAME treatment significantly blunted eNOS-derived<jats:inline-formula><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mrow><mml:msubsup><mml:mi>O</mml:mi><mml:mn>2</mml:mn><mml:mrow><mml:mo>•</mml:mo><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:mrow></mml:mrow></mml:math></jats:inline-formula>generation but did not modify eNOS phosphorylation or monomerization.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>MG triggers eNOS uncoupling and hypophosphorylation in EA.hy926 endothelial cells associated with<jats:inline-formula><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mrow><mml:msubsup><mml:mi>O</mml:mi><mml:mn>2</mml:mn><mml:mrow><mml:mo>•</mml:mo><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:mrow></mml:mrow></mml:math></jats:inline-formula>generation and biopterin depletion. The observed effects of the glycolysis metabolite MG presumably account, at least in part, for endothelial dysfunction in diabetes.</jats:p></jats:sec>