Determinants of shear stress-stimulated endothelial nitric oxide production assessed in real-time by 4,5-diaminofluorescein fluorescence.
The extremely short biological half-life of endothelial-derived nitric oxide (NO) has impeded real-time measurements of NO synthesis. We used the membrane-permeable fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA) to study determinants of NO synthesis in bovine aortic endothelial cells (BAECs). A step increase in shear stress (SS) from 0.3 to 3.4 dyne/cm(2) triggered an increase in DAF-2 fluorescence starting 3.0 +/- 0.5 min after the flow rise and peaking at 44.7 +/- 7.2 min. This was abolished by intracellular Ca(2+) chelation, but was unaffected by blocking extracellular Ca(2+) influx or by inhibiting SS-related changes in intracellular pH. The increase in DAF-2 fluorescence occurred significantly earlier in BAECs transfected with either superoxide dismutase (SOD) or catalase (CAT), indicating concomitant reactive oxygen species (ROS) generation by SS and "competition" between ROS- and DAF-2-NO interactions. These data provide novel insights into several NO signaling determinants and reveal that DAF-2 can assess real-time SS-stimulated NO synthesis in endothelial cells. This should facilitate the analysis of NO-signaling pathways.
Qiu
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Department of Medicine, Johns Hopkins Hospital, Baltimore, Maryland 21224, USA.
Kass
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Hu
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Ziegelstein
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D000818:Animals; D002118:Calcium / Q000502:physiology; D002374:Catalase / Q000235:genetics; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D019793:Fluorescein / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000737:chemistry*; D007700:Kinetics; D009569:Nitric Oxide / Q000096:biosynthesis*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013314:Stress, Mechanical; D013482:Superoxide Dismutase / Q000235:genetics; D014162:Transfection
C111205:4,5-diaminofluorescein; D007202:Indicators and Reagents; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D002374:Catalase; D013482:Superoxide Dismutase; D002118:Calcium; D019793:Fluorescein