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Fig. 4. NO donors increase endocytosis by augmenting dynamin GTPase activity. (A,B) BAEC were preincubated with vehicle or the NO donor GSNO for 20 minutes and then further incubated with fluorescent ligands [fluorescent 488 conjugated transferrin (Tfn) or BODIPY C5-lactosylceramide (LacCer)] for 10 minutes and analyzed by laser scanning cytometry and confocal fluorescence microscopy. Tfn uptake was increased in cells incubated with GSNO as compared with vehicle. Upper panels show representative cell captured by confocal microscopy and lower panels show quantitation by laser scanning cytometry (control R1=6%, GSNO R1=15%). LacCer uptake was also increased in cells incubated with GSNO (control R1=13%, GSNO R1=55%). (C) BAEC were transfected with wild-type dynamin virus or AdK44A prior to GSNO, and Tfn uptake was assayed. AdK44A transduction of EC reduced Tfn uptake and blocked NO-induced increase of Tfn uptake (left: control R1=27%; middle: K44A R1=17%; right: K44A+GSNO R1=18%). (D) Dynamin GTPase activity was measured by thin-layer chromatographic detection of GDP from GTP using recombinant GST-dynamin and a representative experiment is shown. Dynamin incubated with GSNO resulted in an increase in GDP and depletion of GTP, indicating activation of GTPase activity (lane 4). Increased cold GTP competes the conversion from ³²P-labeled GTP to GDP (lanes 5-8). Densitometric analysis of the experiment is shown in the lower panel. This experiment was repeated three times with similar results.

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