QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 22 July 2003
doi: 10.1242/jcs.00664

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chatterjee, S. Articles by Shah, V. Search for Related Content PubMed PubMed Citation Articles by Chatterjee, S. Articles by Shah, V. Social Bookmarking                         What's this?

Inhibition of GTP-dependent vesicle trafficking impairs internalization of plasmalemmal eNOS and cellular nitric oxide production

¹ GI Research Unit, Department of Physiology, and Tumor Biology Program, Mayo Clinic, Rochester, MN 55905, USA
² Division of Cardiovascular Disease, and Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905, USA

View larger version (14K): [in a new window]   Fig. 1. BK stimulates cellular NO production. BK-mediated cellular NO production was measured from eNOS-GFP ECV 304 cells and BAEC using the cell membrane-impermeable and -permeable fluorescent probes, DAF-2 and DAF-2DA, respectively. Cells were cultured in 12-well plates for 24 hours and then culture medium was replaced with PBS containing L-arginine (0.1 mM) or alternatively, L-NAME (1 mM) for 30 minutes, loaded with either DAF-2 or DAF-2DA, then stimulated with BK (10 µM). Relative NO production was measured by fluorimetry from the media for DAF-2 experiments and alternatively assessed by quantitation of fluorescence intensity from captured microscopic images for DAF-2DA experiments. eNOS-GFP ECV 304 cells preincubated with L-arginine produced NO in a time-dependent manner after BK stimulation, as assessed by increased DAF-2 fluorescence in the medium (left panel), as do BAEC, as assessed by increased intracellular DAF-2DA fluorescence (right panel) (n=10 selected areas from each micrograph from 3 independent experiments; *P<0.05 compared to 5 minutes). In both cell types, BK stimulation of NO production was markedly inhibited by L-NAME preincubation (1 mM) (n=3 independent DAF-2 experiments each performed in duplicate; #P<0.05 compared to L-NAME).

View larger version (14K): [in a new window]   Fig. 2. BK promotes eNOS redistribution into a Triton X-100-insoluble fraction. eNOS-GFP ECV 304 cells were incubated with BK (10 µM) or vehicle for 10 minutes then fractionated into cytosol and membrane fractions. Membrane fractions were further fractionated into 0.5% Triton X-100-soluble (TS) and -insoluble fractions (TIF). eNOS protein abundance was assessed in fractions by western blot analysis. While BK stimulation did not influence the distribution of eNOS in membrane (memb) and cytosolic (cytosol) fractions (left panel), BK stimulation was associated with an enrichment of eNOS within the TIF component of the membrane fraction (right panel). Control experiments demonstrate that caveolin protein levels (cav) are enriched in membrane fractions compared to cytosol, and TIF compared to TS (lower panel). Each panel is representative of three separate experiments.

View larger version (56K): [in a new window]   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).

View larger version (36K): [in a new window]   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).

View larger version (14K): [in a new window]   Fig. 5. Redistribution of eNOS-GFP in response to BK stimulation is abrogated by overexpression of the dominant negative dynamin-2 mutant, K44A. (A) To confirm protein separation by buoyant density gradient centrifugation, equal amounts of protein from each fraction were analyzed by SDS-PAGE and western blotting using caveolin pAb, and ß-COP pAb. Enrichment of caveolin, a marker of low buoyant density cellular membranes, was detected in the early fractions (#2-4), while ß-COP, a marker coat protein of Golgi and trans-Golgi, was enriched in the higher buoyant density fractions (#5-7). (B) eNOS-GFP ECV 304 cells were cotransfected with pcDNA V5 empty vector (E) or alternatively a V5 epitope-tagged dyn-2 K44A construct (K). 24 hours later, cells were stimulated with BK (10 µM for 10 minutes; +) or sham treatment (-). Four 100 mm dishes of cells from each experimental group were then prepared for sucrose gradient subcellular fractionation. Equal volumes of each fraction were analyzed for eNOS and V5-K44A expression by SDS-PAGE and western blot analysis using eNOS mAb and V5 mAb, respectively. As seen in the rectangular box in Fig. 5B, in cells transfected with empty vector (E), stimulation with BK (+), was associated with an enrichment of eNOS protein levels in the early fractions of the gradient as compared to cells in the absence of BK stimulation [see eNOS signal in lanes labeled E in fractions #1-4; (-) vs (+)]. In cells transfected with V5-K44A, the relative enrichment of eNOS in early fractions in cells after BK stimulation was no longer apparent [see eNOS signal in lanes labeled (K) in fractions #1-4; (-) vs (+)]. No major changes were observed in the distribution of eNOS in the heavy fractions in response to the various experimental conditions and Coommassie staining of SDS-PAGE gels demonstrated similar protein loading amongst the experimental groups within each fraction (not shown). Overexpression of K44A was confirmed by the detection of a 90 kDa band in total cell lysates from dishes transfected with the vector encoding V5-K44A, and overexpression did not influence total eNOS protein levels from cell lysates (see eNOS and V5 western blot bands under lanes labeled lysate). All experiments presented here were performed three times independently, with similar results.

View larger version (31K): [in a new window]   Fig. 6. GTP--s and dominant negative dyn-2 K44A reduce BK-dependent, L-NAME-inhibited NO production from cells. (A) Digitonin (1 µM)-permeabilized BAEC were coincubated with GTP--S (10 µM) or vehicle for 5 minutes. After washing, cells were loaded with the NO fluorescent probe DAF-2DA and stimulated with 10 µM BK in the presence or absence of L-NAME. Time series photographs, taken to observe the temporal effects of BK on NO production in each experimental group, demonstrated an increase in intracellular DAF-2DA fluorescence intensity in cells treated with BK (top panel). The increase in fluorescence intensity in response to BK was markedly abrogated in cells treated with GTP--S (middle panel), or with both GTP--S and L-NAME (lower panel). The graph below, depicts the quantified fluorescence intensity over time within each group and shows a significant abrogation in NO production in BK-stimulated cells incubated with GTP--S or both GTP--S and L-NAME as compared to BK stimulation alone. Data are representative of three independent experiments with similar results. (B) eNOS-GFP ECV 304 cells were transfected with vectors encoding V5-K44A, a dominant negative mutant of dynamin-2, or empty pcDNA3.1 vector, then serum starved for 24 hours prior to experiments. Cells were stimulated with BK or vehicle in the presence of L-arginine or L-NAME and NO release was assessed from the media using the NO probe DAF-2. After collection of media for fluorimetry, cell lysates were prepared for western blot analysis using V5 mAb, dyn-2 pAb, and eNOS mAb, as well as protein assay. In cells transfected with empty vector, BK stimulated a 2-fold increase in NO production, which was entirely inhibited in the presence of the NOS inhibitor L-NAME. Overexpression of dyn-2 K44A markedly abrogated BK stimulated NO production in the presence and absence of L-NAME. (Closed bars: 0 µM BK; open bars: 10 µM BK; n=5 independent experiments each measured in duplicate). The control immunoblot panels demonstrate overexpression of V5 epitope-tagged dyn-2 using antibodies for V5 as well as dyn-2, and similar levels of eNOS protein between the experimental groups. (C) eNOS-GFP ECV 304 cells were transfected as described above for DAF-2 fluorimetry experiments. Samples of media were collected 20 minutes after BK stimulation and analyzed for nitrite levels using NO-specific chemiluminescence. In cells transfected with empty vector, L-NAME-inhibited NO production was increased by approximately twofold in cells stimulated with BK as compared to control cells (n=3 independent experiments each analyzed with duplicate samples; *P<0.05 compared to other groups). In cells transfected with dyn-2 K44A, no prominent increase in L-NAME inhibited, BK-mediated NO production was detected.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter