Fig. 3. 20-HETE increases dichlorofluorescein (DCFH) fluorescence.
Similar to experiments with DHE, BPAECs were preincubated with 5 μM DCF for 20 min and then imaged for semiquantitation of fluorescent values using MetaMorph 5 min after application of 1 μM 20-HETE (diagonal crossed bars) or vehicle. More than 60 cells for each experimental condition were imaged and analyzed for these experiments. Representative images appear on the left with averaged values shown in bar graphs on the right. Application of ethanol vehicle alone had no effect on DCF fluorescence (open bars), whereas 20-HETE (diagonal cross hatched bars) increased these values 5 min after the addition of the lipid to the bath. Pretreatment with 500 U PEG catalase (PEG-cat; cat. no. C-4963, 17,600 U/mg solid, 1 unit decomposed 1 μmol of H2O2/min at pH 7.0 at 25°C, whereas the H2O2 concentration falls from 10.3 to 9.2 mM) effectively blocked 20-HETE-induced DCF fluorescence. *P < 0.05 relative to vehicle control.