Abstract
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/cm2 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 Ca2+ chelation, but was unaffected by blocking extracellular Ca2+ 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.
Original language | English (US) |
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Pages (from-to) | 328-335 |
Number of pages | 8 |
Journal | Biochemical and Biophysical Research Communications |
Volume | 286 |
Issue number | 2 |
DOIs | |
State | Published - 2001 |
Keywords
- Calcium
- Catalase
- Diaminofluorescein
- Endothelium
- Intracellular pH
- Reactive oxygen species
- Shear stress
- Superoxide dismutase
- Transfection
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology
- Cell Biology