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Mechanistic study of nitric oxide bioavailability in hyperglycemic HUVEC's using DAF‐FM‐DA
Author(s) -
Rogers Steven C.,
Kavdia Mahendra
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.785.14
Subject(s) - apocynin , nadph oxidase , nitric oxide , chemistry , superoxide , superoxide dismutase , oxidative stress , bioavailability , oxidase test , pharmacology , biochemistry , intracellular , enzyme , biology , organic chemistry
Decreased nitric oxide (NO) bioavailability is implicated in a number of disease pathologies. Diabetes is shown to disrupt the vascular homeostasis of NO and superoxide, leading to decreased NO presence and increased oxidative stress. However, relative contribution of individual biochemical pathways responsible for reduced NO availability are not completely known. In this study, we mechanistically analyzed the contribution of NADPH oxidase and NOS on intracellular NO concentrations in hyperglycemic HUVECs. DAF‐FM diacetate fluorescence was used for NO measurements using a microplate reader and further analyzed using fluorescence microscopy. We also tested the impact of inhibitors and antioxidants such as L‐NAME, apocynin, and superoxide dismutase. Hyperglycemic cells showed a decrease in fluorescence intensity over that of normal glucose. Treatment of apocynin and SOD increased NO levels in hyperglycemic cells, whereas the treatment of L‐NAME caused a decrease in NO. Combined treatment of L‐NAME and apocynin had a fluorescence increase over that of the L‐NAME individual treatment. Results show that uncoupling of eNOS leads to dramatically reduced nitric oxide presence. The data also shows that excessive NADPH oxidase activity leads to significant decrease in nitric oxide bioavailability but can be restored through inhibition of NADPH oxidase. Supported by: AHA 0530050N and NIH R01 HL084337