eNOS is active at motility-associated structures of human endothelial cells, where it colocalizes with MT1-MMP. (A) Subcellular location of eNOS (red) and MT1-MMP (green) was analyzed by immunofluorescence double-staining of subconfluent HUVECs. The merged image reveals colocalization (yellow); arrows indicate colocalization at membrane protrusions. (B) Migrating HUVECs were preloaded for 30 minutes with the NO probe DAF-FM, and NO production was clearly visible at cellular protrusions (top). The lower figure shows representative real-time capture (n = 4) of DAF-FM fluorescence at the lamellipodia of a migrating EC. The location of NO production was recorded over 6 minutes at lamellipodia in migrating ECs at 20-second intervals; t = 0 indicates the beginning of recording. Note the appearance and disappearance of NO production at lamellipodia during the recorded period (bottom, arrowheads). (C) HUVECs were left untreated or treated with L-NAME for 1 hour. Migration was initiated by disrupting monolayers (“In situ detection and measurement of NO production”). NO production was measured as the accumulation of NO_2^- released into the culture medium over 30 minutes by resting (confluent) or migrating (wound-healing) HUVECs. The chart shows the arithmetic mean plus or minus the standard deviation (SD) of the fold induction (FI) over nitrite release from 3 independent experiments run in duplicate. The absolute value corresponding to FI = 1 was 307 nM. *P < .04.