期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 286, 期 2, 页码 328-335出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1006/bbrc.2001.5401
关键词
shear stress; endothelium; diaminofluorescein; calcium; intracellular pH; reactive oxygen species; superoxide dismutase; catalase; transfection
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 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. (C) 2001 Academic Press.
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