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CARDIOVASCULAR

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

Smooth Muscle Research Group, Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta, Canada

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.

Address correspondence to: Dr. Andrew Braun, Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, 3330 Hospital Drive, N.W., Calgary, Alberta T2N 4N1, Canada. E-mail: abraun{at}ucalgary.ca

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				J.-Z. Sheng, F. Arshad, J. E. Braun, and A. P. Braun Estrogen and the Ca2+-mobilizing agonist ATP evoke acute NO synthesis via distinct pathways in an individual human vascular endothelium-derived cell Am J Physiol Cell Physiol, June 1, 2008; 294(6): C1531 - C1541. [Abstract] [Full Text] [PDF]

				J.-Z. Sheng and A. P. Braun Small- and intermediate-conductance Ca2+-activated K+ channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells Am J Physiol Cell Physiol, July 1, 2007; 293(1): C458 - C467. [Abstract] [Full Text] [PDF]