eNOS phosphorylation increased intracellular ROS generation and decreased cell viability in BH4-depleted cells.

<p>(A) HEK 293 cells were treated with DMSO or 10mM DAHP for 24 hours to deplete BH4, then transfected with either control vector or WT eNOS plasmid DNA overnight. The transfected cells were incubated with DCF-DA and VEGF as indicated. The fluorescence density was recorded by fluorescent microscopy described in Materials and Methods. (A). Representative fluorescent images are shown. There was no DCF-DA signal in untreated eNOS null HEK 293 cells (first panel, 293 control) or eNOS HEK 293 cells (Fourth panel, eNOS 293 control). A very weak DCF-DA signal was observed in eNOS null HEK 293 in presence of DAHP alone, or with DAHP and VEGF (50ng/mL) together (second panel and third panel, respectively). However, following depletion of BH4 by 10mM DAHP for 48 hours, a prominent DCF-DA signal was observed in eNOS 293 cells (fifth panel). A stronger DCF-DA signal was observed in VEGF-stimulated and BH4-depleted eNOS 293 cells (sixth panel). (B) VEGF-mediated eNOS phosphorylation resulted a significant ROS increase in eNOS overexpressing HEK 293 cells (n = 5 fields, *, <i>p</i> < 0.05). (C) eNOS phosphorylation decreased cellular viability in BH4-depleted cells. MTT assays were used to evaluate cellular injury induced by eNOS phosphorylation following BH4 depletion. For eNOS overexpressing HEK 293 cells (Black bars), cellular viability was significantly decreased in DAHP treated cells compared with untreated control cells (HEK 293 eNOS +DAHP vs. HEK293 eNOS control, *, <i>p</i> < 0.01). 50ng/mL VEGF caused further decrease of cell viability in eNOS overexpressing HEK293 cells treated with 10mM DAHP (VEGF plus DAHP vs. DAHP alone in HEK 293 eNOS cells, #, <i>p</i> < 0.01).