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Comparative Study
. 2016 Mar 15;11(3):e0151556.
doi: 10.1371/journal.pone.0151556. eCollection 2016.

Activation of Endothelial Nitric Oxide (eNOS) Occurs through Different Membrane Domains in Endothelial Cells

Jason Tran  1   2 , Astrid Magenau  1   2 , Macarena Rodriguez  1   2 , Carles Rentero  3 , Teresa Royo  3   4 , Carlos Enrich  3   4 , Shane R Thomas  5 , Thomas Grewal  6 , Katharina Gaus  1   2
Affiliations
Comparative Study

Activation of Endothelial Nitric Oxide (eNOS) Occurs through Different Membrane Domains in Endothelial Cells

Jason Tran et al. PLoS One. .

Abstract

Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS) and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC) with cholesterol and the oxysterol 7-ketocholesterol (7KC). Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1) colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL)-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF)-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Immunofluorescence of sterol-manipulated BAEC.
(A) BAEC plated on gelatin-coated coverslips were enriched with 40 μM Chol, 20 μM 7KC or a mixture of 20 μM Chol and 20 μM 7KC (Ch:7KC), fixed and immunostained with Cav1-Alexafluor 647 and eNOSIII-Alexafluor 488 and visualized by confocal fluorescence microscopy. Scale bar: 20 μM. (B) Pearson’s correlation of Cav1 and eNOS co-localization in confocal images. (C-F) Fluorescence intensity of eNOS (D-C) and Cav1 (E-F) in the plasma membrane (C, E) and in intracellular regions (D, F) in confocal images. The plasma membrane was defined as the region ~1.2 μm from the cell periphery and intracellular regions as adjacent to the plasma membrane regions. In C-F, each symbol represents one (merged) image and horizontal bars indicate means. Significant differences to control cells were tested with one-way ANOVA.
Fig 2
Fig 2. Effect of sterol enrichment on BAEC membrane order.
BAEC were enriched with 40 μM Chol, 20 μM 7KC or a mixture of 20 μM Chol and 20 μM 7KC (Ch:7KC) and stained with 5 μM Laurdan followed by fixation and immunostaining for Cav1. (A) Confocal image of Cav1-Cy3 staining (top row), Laurdan GP image of the same focal depth (middle row) and masked GP images showing only the GP image of Cav1-positiv membrane areas (bottom row). GP images were pseudo-coloured according to the colour scale shown in the first GP image. Blue colour indicates GP values of 0.3 and red colour indicates GP values of 1.0. Scale bar = 20 μm. (B) Total plasma membrane GP values were determined from GP images recorded at high magnification. All sterol enrichment conditions were significantly different (P<0.001) to control cells. (C) Cav1-positive membrane GP values were determined using the masked GP image. Cholesterol and Chol:7KC enrichment increased membrane order significantly (P<0.001), while 7KC-treatment also increased membrane order compared to control (P<0.01). Data are from three independent experiments with a total of 60 cells. In B-C, each symbol is one image, typically with one cell; horizontal lines indicate means. Significances were tested with one-way ANOVA.
Fig 3
Fig 3. Caveolae quantification in control and sterol-enriched BAEC.
BAEC were enriched with 40 μM Chol, 20 μM 7KC or a mixture of both 20 μM Chol and 20 μM 7KC (Ch:7KC). Cells were fixed and embedded in Spur resin, processed for TEM, and imaged. The images were connected using the plug-in Mosaic J in Image J. Representative TEM images of (A) control, (B) Chol, (C) Ch:7KC, and (D) 7KC-enriched cells. In (D), images were merged and a zoomed region is shown with caveolae visible in the membrane. Images are representative of >20 cells per condition. Scale bar = 0.5 μm (A-C) and 1 μm (D). (E) Caveolae was quantified along the plasma membrane to obtain the number of caveolae/μm of cell perimeter. Each symbol represents one cell and several images. Data were from 5 independent experiments. Horizontal lines indicate means. No significant difference was observed between control cells and sterol enriched cells (tested with one-way ANOVA).
Fig 4
Fig 4. Quantification of Cav1 distributions by PALM and dSTORM.
BAEC were transfected with Cav1-mEOS2 for PALM imaging (A, C-D) or immuno-stained with Cav1-Alexa Fluor 647 for dSTORM imaging (B, E-F). BAEC were left untreated (control) or treated with sterols as indicated, fixed and imaged by PALM and dSTORM. (A-B) PALM (A) and dSTORM (B) TIRF images of Cav1 in the membrane of BAEC (scale bar = 5 μm) with the white square indicating the zoomed region (scale bar = 0.5 μm). Each black dot represents one detected Cav1 molecule. (C-F) Ripley’s K-function plots (C, E) and quantification (D,F) of Cav1 distribution of PALM (C-D) and dSTORM (E-F) images. The L(r)-r reports the degree of non-randomness in point patterns on the radial length scale, r, relative to a random distribution (indicated by the dotted lines). In C and E, Ripley’s K-function plots of control (green), cholesterol- (red), Chol:7KC- (orange) and 7KC-treated cells (blue) are shown. In D and F, the maximum L(r)-r values reflect the maximum degree of clustering. Each symbol is one image region; horizontal bars reflect means. Significances were calculated with one-way ANOVA (no significant differences were found in F).
Fig 5
Fig 5. eNOS activity of sterol-manipulated BAEC in response to various stimuli.
BAEC were enriched with 40 μM Chol, 20 μM 7KC or a mixture of both (20 μM Chol:20 μM 7KC). Cells were left untreated (control, A) or stimulated with 25 ng/ml VEGF for 5 min (B), 50 μg/ml HDL for 20 min (C) or with 10 dynes/cm2 shear stress for 5 min (D). Data is reported as the extent of conversion of [3H]-L-arginine to [3H]-L-citrulline that is sensitive to pre-treatment of endothelial cells for 30 min with 1 mM L-NAME and is expressed as dpm/106 cells. Data were normalised to control and are expressed as means ± SEM from at least seven independent experiments performed in triplicate. Significance compared to control was calculated by one-way ANOVA and one asterisk indicates P<0.05.
Fig 6
Fig 6. eNOS phosphorylation of sterol-manipulated BAEC in response to various stimuli.
BAEC were enriched were treated as above and then either not stimulated (A) or stimulated with VEGF (25 ng/ml) for 5 min (B), 50 μg/ml HDL for 20 min (C) or stimulated with 10 dynes/cm2 shear stress for 5 min (D). Cells were lysed and equal protein amounts analysed by immunoblotting with antibodies against phospho-eNOSSer1179 and total eNOS. Phosphorylation was measured with densitometry of phosphorylated protein divided by the total protein and normalised to control conditions. Data represent means ± SEM from six independent experiments. Significance compared to control was calculated by one-way ANOVA and is indicated by one asterisk reflecting P<0.05 or two asterisks indicating P<0.01.
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This work was supported by the Australian Research Council (ARC) and National Health and Medical Research Council (NHMRC) of Australia (Program grant 1037320). TG received support from The University of Sydney, Australia (U1758, U7007). CE received support from the Ministerio de Economía y Competitividad (MINECO; BFU2012-36272, CSD2009-00016) and Fundació Marató TV3 (PI042182) (Spain). CR received a post-doctoral fellowship (CSD2009-00016) from CONSOLIDER-INGENIO (MINECO) research program and support from Fundació Marató TV3. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.