Fig. 1. BK stimulates cellular NO production. BK-mediated cellular NO production was measured from eNOS-GFP ECV 304 cells and BAEC using the cell membrane-impermeable and -permeable fluorescent probes, DAF-2 and DAF-2DA, respectively. Cells were cultured in 12-well plates for 24 hours and then culture medium was replaced with PBS containing L-arginine (0.1 mM) or alternatively, L-NAME (1 mM) for 30 minutes, loaded with either DAF-2 or DAF-2DA, then stimulated with BK (10 µM). Relative NO production was measured by fluorimetry from the media for DAF-2 experiments and alternatively assessed by quantitation of fluorescence intensity from captured microscopic images for DAF-2DA experiments. eNOS-GFP ECV 304 cells preincubated with L-arginine produced NO in a time-dependent manner after BK stimulation, as assessed by increased DAF-2 fluorescence in the medium (left panel), as do BAEC, as assessed by increased intracellular DAF-2DA fluorescence (right panel) (n=10 selected areas from each micrograph from 3 independent experiments; ^*P<0.05 compared to 5 minutes). In both cell types, BK stimulation of NO production was markedly inhibited by L-NAME preincubation (1 mM) (n=3 independent DAF-2 experiments each performed in duplicate; ^#P<0.05 compared to L-NAME).

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