Received for publication April 14, 2005.
Revised August 8, 2005.
Accepted for publication August 8, 2005.

DAF-FM Diacetate Detects Impairment of Agonist-stimulated Nitric Oxide Synthesis by Elevated Glucose in Human Vascular Endothelial Cells: Reversal by Vitamin C and L-Sepiapterin

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, 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate to examine directly the consequences of elevated glucose on agonist-evoked NO synthesis in cultured human vascular endothelial cells. Exposure of cells for 5-7 days to high (20 mM) external glucose markedly reduced NO production in response to ATP, histamine or the Ca ionophore calcimycin A23187, compared to 5 and 10 mM glucose concentrations. However, high glucose did not affect agonist-evoked elevations in cytosolic free calcium, as monitored by Fluo-3. 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. Similarly, impaired NO production in high glucose-treated cells was largely reversed by exposure (~3 hr) to superoxide dismutase. Cellular levels of eNOS protein were unaltered by elevated glucose treatment, and no further change was observed following addition of vitamin C and L-sepiapterin. Taken together, the results of our study serve to explain directly 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.

Key words: diabetes, endothelium, fluorescence, glucose, human, nitric oxide

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