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DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein) Diacetate Detects Impairment of Agonist-Stimulated Nitric Oxide Synthesis by Elevated Glucose in Human Vascular Endothelial Cells: Reversal by Vitamin C and L-sepiapterin

DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein) Diacetate Detects Impairment of Agonist-Stimulated Nitric Oxide Synthesis by Elevated Glucose in Human Vascular Endothelial Cells: Reversal by Vitamin C and L-sepiapterin

Jan-Zhong Sheng, Dianna Wang, Andrew P Braun

J Pharmacol Exp Ther. 2005 Nov;315(2):931-40.

PMID: 16093274

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 microM) and L-sepiapterin (20 microM) for approximately 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 ( approximately 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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP254109201-A	DAF-FM		254109-20-1	Price

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