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Long Y, Yan J, Luo S, Liu Z, Xia Y. Measurement of O-GlcNAcylated endothelial nitric oxide synthase by using 2',5'-ADP-Sepharose pull-down assay. Analytical biochemistry 2017 537 28844813
Abstract:
Endothelial nitric oxide synthase (eNOS) plays central roles in cardiovascular regulation and disease. eNOS function is critically affected by O-linked N-acetylglucosamine (O-GlcNAc) modification. The present method for measuring O-GlcNAcylated eNOS relies on immunoprecipitation. Such method exhibits low detection efficiency and is also costly. We here report a simplified assay by employing the high binding affinity of eNOS with the 2',5'-ADP-Sepharose resins. Together with the O-GlcNAc antibody, this assay readily allows the detection of O-GlcNAcylated eNOS in both cultured endothelial cells and rat vascular tissues. By using this assay, we demonstrate that eNOS O-GlcNAcylation is markedly elevated in the vessels of diabetic rats. Thus, a 2',5'-ADP-Sepharose-based pull-down assay is developed to measure O-GlcNAcylated eNOS. This assay is simple and efficient in detecting O-GlcNAcylated eNOS in cultured cells and animal tissues under both normal and disease conditions.
O-GlcNAc proteins:
NOS3, NOS3
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Lima VV, Giachini FR, Choi H, Carneiro FS, Carneiro ZN, Fortes ZB, Carvalho MH, Webb RC, Tostes RC. Impaired vasodilator activity in deoxycorticosterone acetate-salt hypertension is associated with increased protein O-GlcNAcylation. Hypertension (Dallas, Tex. : 1979) 2009 53(2) 19139380
Abstract:
Hyperglycemia, which increases O-linked beta-N-acetylglucosamine (O-GlcNAc) proteins, leads to changes in vascular reactivity. Because vascular dysfunction is a key feature of arterial hypertension, we hypothesized that vessels from deoxycorticosterone acetate and salt (DOCA-salt) rats exhibit increased O-GlcNAc proteins, which is associated with increased reactivity to constrictor stimuli. Aortas from DOCA rats exhibited increased contraction to phenylephrine (E(max) [mN]=17.6+/-4 versus 10.7+/-2 control; n=6) and decreased relaxation to acetylcholine (47.6+/-6% versus 73.2+/-10% control; n=8) versus arteries from uninephrectomized rats. O-GlcNAc protein content was increased in aortas from DOCA rats (arbitrary units=3.8+/-0.3 versus 2.3+/-0.3 control; n=5). PugNAc (O-GlcNAcase inhibitor; 100 micromol/L; 24 hours) increased vascular O-GlcNAc proteins, augmented phenylephrine vascular reactivity (18.2+/-2 versus 10.7+/-3 vehicle; n=6), and decreased acetylcholine dilation in uninephrectomized (41.4+/-6 versus 73.2+/-3 vehicle; n=6) but not in DOCA rats (phenylephrine, 16.5+/-3 versus 18.6+/-3 vehicle, n=6; acetylcholine, 44.7+/-8 versus 47.6+/-7 vehicle, n=6). PugNAc did not change total vascular endothelial nitric oxide synthase levels, but reduced endothelial nitric oxide synthase(Ser-1177) and Akt(Ser-473) phosphorylation (P<0.05). Aortas from DOCA rats also exhibited decreased levels of endothelial nitric oxide synthase(Ser-1177) and Akt(Ser-473) (P<0.05) but no changes in total endothelial nitric oxide synthase or Akt. Vascular O-GlcNAc-modified endothelial nitric oxide synthase was increased in DOCA rats. Blood glucose was similar in DOCA and uninephrectomized rats. Expression of O-GlcNAc transferase, glutamine:fructose-6-phosphate amidotransferase, and O-GlcNAcase, enzymes that directly modulate O-GlcNAcylation, was decreased in arteries from DOCA rats (P<0.05). This is the first study showing that O-GlcNAcylation modulates vascular reactivity in normoglycemic conditions and that vascular O-GlcNAc proteins are increased in DOCA-salt hypertension. Modulation of increased vascular O-GlcNAcylation may represent a novel therapeutic approach in mineralocorticoid hypertension.
O-GlcNAc proteins:
NOS3
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Musicki B, Kramer MF, Becker RE, Burnett AL. Inactivation of phosphorylated endothelial nitric oxide synthase (Ser-1177) by O-GlcNAc in diabetes-associated erectile dysfunction. Proceedings of the National Academy of Sciences of the United States of America 2005 102(33) 16085713
Abstract:
Impaired endothelial nitric oxide synthase (eNOS) function is associated with erectile dysfunction in diabetes mellitus, but the exact molecular basis for the eNOS defect in the diabetic penis remains unclear. We investigated whether hyperglycemia increases O-GlcNAc modification of eNOS in the penis, preventing phosphorylation at the primary positive regulatory site on the enzyme and hampering mechanisms of the erectile response. Type I diabetes mellitus was induced in male rats by alloxan (140 mg/kg, i.p.). After 5 wk, the diabetic rat penis exhibited increased O-GlcNAc modification of eNOS and decreased eNOS phosphorylation at Ser-1177 at baseline compared with the control rat penis; eNOS phosphorylation at Thr-495, Ser-615, and Ser-633 was not affected. In addition, eNOS phosphorylation at Ser-1177 was impaired in the diabetic rat penis in response to penile blood flow (shear stress) elicited by electrical stimulation of the cavernous nerve (ES) and to recombinant human VEGF165. Phosphorylation of Akt, a mediator of shear stress-induced eNOS phosphorylation at Ser-1177, was decreased in the diabetic penis at baseline, but it was restored by ES. Erectile response to shear stress elicited by ES and to VEGF was decreased in diabetic compared with control rats. This work demonstrates that eNOS inactivation occurs in the diabetic penis by a glycosylation mechanism specifically at Ser-1177, by which the enzyme is rendered incapable of activation by fluid shear stress stimuli and VEGF signaling. In vivo penile erection paradigm supports the physiologic relevance of O-GlcNAc modification in vascular disorders associated with diabetes.
O-GlcNAc proteins:
NOS3
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