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.
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.
Sheng
Jan-Zhong
JZ
Smooth Muscle Research Group, Department of Pharmacology and Therapeutics, University of Calgary, Alberta, Canada.
Wang
Dianna
D
Braun
Andrew P
AP
D000255:Adenosine Triphosphate / Q000378:metabolism; D001205:Ascorbic Acid / Q000494:pharmacology*; D001682:Biological Availability; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D005452:Fluoresceins; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D011622:Pterins / Q000494:pharmacology*; D014815:Vitamins / Q000494:pharmacology*
C503301:4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; D005452:Fluoresceins; D011622:Pterins; D014815:Vitamins; D009569:Nitric Oxide; D000255:Adenosine Triphosphate; C016727:sepiapterin; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D005947:Glucose; D001205:Ascorbic Acid; D002118:Calcium