title: Activation of Endothelial Nitric Oxide (eNOS) Occurs through Different Membrane Domains in Endothelial Cells.
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.
authors.affiliation: EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, Australia.|ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, Australia.
authors.forename: Jason
authors.initials: J
authors.lastname: Tran
authors.affiliation: EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, Australia.|ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, Australia.
authors.forename: Astrid
authors.initials: A
authors.lastname: Magenau
authors.affiliation: EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, Australia.|ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, Australia.
authors.forename: Macarena
authors.initials: M
authors.lastname: Rodriguez
authors.affiliation: Departament de Biologia Cel·lular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain.
authors.forename: Carles
authors.initials: C
authors.lastname: Rentero
authors.affiliation: Departament de Biologia Cel·lular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain.|Centre de Recerca Biomèdica CELLEX, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain.
authors.forename: Teresa
authors.initials: T
authors.lastname: Royo
authors.affiliation: Departament de Biologia Cel·lular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain.|Centre de Recerca Biomèdica CELLEX, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Enrich
authors.affiliation: School of Medical Sciences, The University of New South Wales, Sydney, Australia.
authors.forename: Shane R
authors.initials: SR
authors.lastname: Thomas
authors.affiliation: Faculty of Pharmacy A15, University of Sydney, Sydney, Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Grewal
authors.affiliation: EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, Australia.|ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, Australia.
authors.forename: Katharina
authors.initials: K
authors.lastname: Gaus
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D002417:Cattle; D021941:Caveolae / Q000502:physiology; D051242:Caveolin 1 / Q000032:analysis; D002460:Cell Line; D002784:Cholesterol / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007653:Ketocholesterols / Q000494:pharmacology; D008075:Lipoproteins, HDL / Q000494:pharmacology; D021962:Membrane Microdomains / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D012212:Rheology; D013314:Stress, Mechanical; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D051242:Caveolin 1; D007653:Ketocholesterols; D008075:Lipoproteins, HDL; D042461:Vascular Endothelial Growth Factor A; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D052250:Nitric Oxide Synthase Type III; C003001:7-ketocholesterol