Chemiluminescence detection of nitric oxide production from rat cerebral cortical endothelial cells in culture.
Cultured brain microvascular endothelial cells have been used to study blood-brain barrier properties in vitro. Among several endothelial derived factors the gaseous free radical nitric oxide (NO) has been shown to regulate both vascular tone and blood-brain barrier permeability. Due to its short half-life and high reactivity with many other molecules, effects of NO have preferentially been investigated using NO synthase inhibitors, NO donors or, more recently, in NO synthase knock-out animals. In the present study we describe a rapid, sensitive and reliable protocol for chemiluminescence detection of NO2-, the stable oxydation product of NO, from the supernatant of rat cortical endothelial cells in culture. After suppression of inducible NOS activity brain endothelial cells showed low basal NO levels. After stimulation with calcium ionophore NO levels increased and were attenuated again by NOS blockade. NO release was also elicited by bradykinin or acetylcholine, albeit to a lesser extent. This protocol might prove useful to investigate calcium-dependent NO formation in cerebral endothelial cells and blood-brain barrier models in vitro. In principle, the same protocol can be applied to virtually any cell type in culture.
Weih
M K
MK
Department of Experimental Neurology, Charité Hospital, Humboldt-University, Schumannstr 20-21, 10098 Berlin, Germany.
Weikert
S
S
Freyer
D
D
Dirnagl
U
U
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000098:blood supply / Q000166:cytology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D008163:Luminescent Measurements; D008833:Microcirculation / Q000502:physiology; D009569:Nitric Oxide / Q000096:biosynthesis*; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results
D009569:Nitric Oxide