PT  - JOURNAL ARTICLE
AU  - Jan-Zhong Sheng
AU  - Dianna Wang
AU  - Andrew P. Braun
TI  - 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 <span class="sc">l</span>-Sepiapterin
AID  - 10.1124/jpet.105.087932
DP  - 2005 Nov 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 931--940
VI  - 315
IP  - 2
4099  - http://jpet.aspetjournals.org/content/315/2/931.short
4100  - http://jpet.aspetjournals.org/content/315/2/931.full
SO  - J Pharmacol Exp Ther2005 Nov 01; 315
AB  - 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. The American Society for Pharmacology and Experimental Therapeutics