Determinants of shear stress-stimulated endothelial nitric oxide production assessed in real-time by 4,5-diaminofluorescein fluorescence

Weiping Qiu, David A. Kass, Qinghua Hu, Roy C. Ziegelstein

Research output: Contribution to journalArticle

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 languageEnglish (US)
Pages (from-to)328-335
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume286
Issue number2
DOIs
StatePublished - Jan 1 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

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