Abstract
Elevated plasma glucose, as commonly seen in types I and II diabetes mellitus, is known to result in endothelial dysfunction, a condition characterized by a loss of nitric oxide (NO)-dependent regulation of vascular tone. In the present study, we have utilized a recently developed NO-sensitive fluorescent dye, DAF-FM (4-amino-5-methylamino-2′,7′-difluorofluorescein) diacetate to directly examine the consequences of elevated glucose on agonist-evoked NO synthesis in cultured human vascular endothelial cells. Exposure of cells for 5 to 7 days to high (20 mM) external glucose markedly reduced NO production in response to ATP, histamine, or the calcium ionophore calcimycin A23187 compared with 5 and 10 mM glucose concentrations. However, high glucose did not affect agonist-evoked elevations in cytosolic-free calcium, as monitored by Fluo-3. The addition of vitamin C (150 μM) and l-sepiapterin (20 μM) for ∼24 h to 20 mM glucose-treated cells improved stimulus-evoked NO synthesis but had no effect on cells exposed to either 5 or 10 mM glucose. Likewise, impaired NO production in high glucose-treated cells was largely reversed by exposure (∼3 h) to superoxide dismutase. Cellular levels of endothelial nitric-oxide synthase protein were unaltered by elevated glucose treatment, and no further change was observed after the addition of vitamin C and l-sepiapterin. Taken together, the results of our study serve to directly explain at the cellular level how glucose-impaired NO production in human endothelial cells may be reversed by agents that are reported clinically to improve endothelium-dependent vasorelaxation in patients.
Footnotes
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This study was supported by an operating grant from the Canadian Institutes of Health Research and an Establishment grant from the Alberta Heritage Foundation for Medical Research (to A.P.B.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.087932.
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ABBREVIATIONS: NO, nitric oxide; DAF-FM, 4-amino-5-methylamino-2′,7′-difluorofluorescein; eNOS, endothelial nitric-oxide synthase; BH4, tetrahydrobiopterin; l-NAME, l-nitro-arginine methyl ester; DMEM, Dulbecco's modified Eagle's medium; AM, acetomethyl ester.
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↵1 These authors contributed equally to this study.
- Received April 13, 2005.
- Accepted August 8, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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