Abstract
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 superoxide 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. 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 enhanced 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 generation but did not modify eNOS phosphorylation or monomerization.ConclusionMG triggers eNOS uncoupling and hypophosphorylation in EA.hy926 endothelial cells associated with generation and biopterin depletion. The observed effects of the glycolysis metabolite MG presumably account, at least in part, for endothelial dysfunction in diabetes.
Highlights
Chronic hyperglycemia fosters endothelial dysfunction that accounts for the pathophysiology of microvascular sequelae in diabetes [1,2]
MG triggers endothelial nitric oxide synthase (eNOS) uncoupling and hypophosphorylation in EA.hy926 endothelial cells associated with O2− generation and biopterin depletion
Treatment of EA.hy926 endothelial cells with MG (50 – 200 μM) for 2, 4, 6 and 8 h respectively, significantly increased O2− production in a concentration- and timedependent manner (Figure 1A). In another series of experiments, we analyzed the effect of biopterin supplementation (BH4, 100 μM; sepiapterin, 20 μM), pharmacological inhibition of NOS using NG-nitro-L-arginine methyl ester (L-NAME) (50 μM) or suppression of ONOO− formation using combined 24-h pretreatment with Tempol (300 μM) and L-NAME (50 μM) on MGinduced O2− production
Summary
Chronic hyperglycemia fosters endothelial dysfunction that accounts for the pathophysiology of microvascular sequelae in diabetes [1,2]. Endothelial nitric oxide synthase (eNOS) is the predominant and constitutively expressed NOS in vascular endothelial cells and catalyzes the reaction for generation of nitric oxide (NO) from L-arginine in the presence of the cofactors tetrahydrobiopterin (BH4) and NADPH [12]. Regulation of eNOS activity is coupled to cytosolic Ca2+ [13]. Alterations of NO balance contribute to the pathophysiology of diabetic complications [15]. Increased levels of the sugar metabolite methylglyoxal (MG) in vivo were shown to participate in the pathophysiology of vascular complications in diabetes. Activity by hypophosphorylation of the enzyme and enhanced monomerization are found in the diabetic milieu, and the regulation of this still remains mechanisms by which MG modulates euNnOdeSf-iansesdo.ciUasteindgfuvanrciotiuosnsphoaf rMmGa-csotilmoguiclaatleadpspurpoeacrohxeisd,ewÀeOe2−luÁcpidroatdeucptuiotant,ive phosphorylation status and eNOS uncoupling in EA.hy926 human endothelial cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
