Methylglyoxal modulates endothelial nitric oxide synthase-associated functions in EA.hy926 endothelial cells.
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 O₂•⁻ 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 N(G)-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.


RESULTS
In EA.hy926 cells, MG treatment significantly enhanced O₂•⁻ 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 O₂•⁻ generation but did not modify eNOS phosphorylation or monomerization.


CONCLUSIONS
MG triggers eNOS uncoupling and hypophosphorylation in EA.hy926 endothelial cells associated with O₂•⁻ generation and biopterin depletion. The observed effects of the glycolysis metabolite MG presumably account, at least in part, for endothelial dysfunction in diabetes.
Department of Pharmacology, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, Canada. lixin.liu@usask.ca.
Yang
Y
Su
Syed M
SM
Qadri
Lingyun
L
Wu
Lixin
L
Liu
D000975:Antioxidants / Q000494:pharmacology; D001708:Biopterins / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism; D010766:Phosphorylation; D055503:Protein Multimerization; D011765:Pyruvaldehyde / Q000633:toxicity*; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D000975:Antioxidants; D004791:Enzyme Inhibitors; D013481:Superoxides; D030421:Peroxynitrous Acid; D001708:Biopterins; C002744:3-nitrotyrosine; D014443:Tyrosine; D011765:Pyruvaldehyde; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III