Fig. 4. Internalization of eNOS in response to BK is inhibited by the non-hydrolyzed GTP analog, GTP--S, but not by inhibitors of clathrin-mediated endocytosis. (A) Digitonin (1 µM)-permeabilized eNOS-GFP ECV304 cells were incubated with vehicle or GTP--S then stimulated with BK or vehicle. Cells were fixed and prepared for confocal microscopy. (Left) In digitonin-treated cells, eNOS protein was detected in a predominantly plasmalemmal and perinuclear location. (Middle) Stimulation of cells with BK (10 µM) was associated with a reduction in plasmalemmal eNOS immunostaining. (Right) Pretreatment with GTP--S abrogated the internalization of plasmalemmal eNOS that was observed with BK stimulation as shown by maintenance of substantive pools of eNOS in the plasma membrane. (B) The graph, generated from data in BAEC, depicts the percentage of cells expressing eNOS in the plasma membrane and in a perinuclear distribution (PM: open bars) compared with cells containing eNOS in a perinuclear distribution only (non-PM: shaded bars), in each of the experimental groups. The percentage of cells with any amount of eNOS in plasma membrane (PM) is diminished after stimulation with BK and the effect of BK is largely abrogated in the presence of GTP--S (n=200 cells in each group from 2 independent experiments; mean±s.e.m.). (C) Under basal conditions, pools of eNOS-GFP are detected in a plasmalemmal and perinuclear distribution in ECV304 cells (image a). In response to BK, internalization of plasmalemmal eNOS-GFP is detected (image b). BK-mediated eNOS-GFP internalization is not abrogated in cells pre-incubated with CP (15 µM) for 10 minutes, prior to 10 µM BK stimulation (image c). eNOS-GFP ECV304 cells were also transfected with vectors encoding myc-AP 180 or empty vector, then stimulated with BK 24 hours later. Cells for study were chosen from the myc-immunofluorescent-positive population. Despite detection of adequate levels of myc protein by western blot (image d, insert), no attenuation in BK-mediated eNOS-GFP internalization was observed (image d).

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