Fig. 3. BK promotes internalization of plasmalemmal eNOS. (A) Subcellular localization of eNOS was assessed by laser scanning confocal microscopy in live eNOS-GFP ECV304 cells, cultured on coverslips. Coverslips were transferred to a Zeiss temperature controlled perfusion system mounted on the microscope stage. (Top panel; BK-), eNOS-GFP was most prominent in plasma membrane and perinuclear regions of the cell, and its distribution was not influenced in the absence of BK during the 12 minutes of the experiment (arrows denote plasmalemmal eNOS). The graph accompanying the top panel quantifies the fluorescence intensity in plasma membrane (PM: solid line with diamonds) and perinuclear region (non-PM: dashed line with squares) in the sequential micrographic images. (Bottom panel; BK+), stimulation of cells with BK (10 µM) triggered a reduction in fluorescence intensity of eNOS-GFP from the plasma membrane (arrows denote distribution of plasmalemmal eNOS). The graph accompanying the bottom panel quantifies a decrease in fluorescence in PM (solid line with diamonds), but not in the perinuclear, non-PM areas (dashed line with squares). The displayed images are representative of 7 independent experiments, and the graphs were generated by compiling the data from 3 cells from each group (mean±s.e.m.). (B) Influence of BK on the distribution of Na⁺-K⁺ ATPase and eNOS-GFP was assessed by immunofluorescence confocal microscopy in fixed ECV304 cells. In the absence of BK stimulation, substantive pools of Na⁺-K⁺ ATPase (top left panel) and eNOS-GFP (top middle panel, arrows denote plasmalemmal eNOS) are detected in the cell periphery, and colocalization of discrete pools of the proteins is detected in portions of the cell periphery (top right panel, arrows denote colocalization in yellow). After BK stimulation, Na⁺-K⁺ ATPase remains in a plasmalemmal distribution (bottom left panel), while substantive pools of eNOS-GFP dissociate from the plasma membrane (bottom middle panel, arrows denote lack of eNOS in plasma membrane) as further evidenced by lack of colocalization with plasmalemmel Na⁺-K⁺ ATPase (lower right). (C) Influence of BK on the distribution of eNOS-GFP in comparison to Golgi 58K protein was assessed by immunofluorescence confocal microscopy in fixed ECV304 cells. In the absence of BK stimulation, substantive pools of 58K protein reside in a perinuclear distribution (top left panel) and pools of eNOS-GFP also reside in a perinuclear distribution (top middle panel, denoted by arrows). Colocalization of the two proteins is detected in a perinuclear Golgi pattern (top right panel, yellow). After BK stimulation of cells, 58K protein remains in a perinuclear distribution (bottom left panel), and the pool of perinuclear eNOS-GFP remains in a similar distribution (bottom middle panel), as further evidenced by maintenance of colocalization of the two proteins in a perinuclear distribution (lower right panel, colocalization denoted in yellow).

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