title: Functional significance of cytosolic endothelial nitric-oxide synthase (eNOS): regulation of hyperpermeability.
abstract: Endothelial NOS (eNOS)-derived NO is a key factor in regulating microvascular permeability. We demonstrated previously that eNOS translocation from the plasma membrane to the cytosol is required for hyperpermeability. Herein, we tested the hypothesis that eNOS activation in the cytosol is necessary for agonist-induced hyperpermeability. To study the fundamental properties of endothelial cell monolayer permeability, we generated ECV-304 cells that stably express cDNA constructs targeting eNOS to the cytosol or plasma membrane. eNOS-transfected ECV-304 cells recapitulate the eNOS translocation and permeability properties of postcapillary venular endothelial cells (Sánchez, F. A., Rana, R., Kim, D. D., Iwahashi, T., Zheng, R., Lal, B. K., Gordon, D. M., Meininger, C. J., and Durán, W. N. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 6849-6853). We used platelet-activating factor (PAF) as a proinflammatory agonist. PAF activated eNOS by increasing phosphorylation of Ser-1177 and inducing dephosphorylation of Thr-495, increasing NO production, and elevating permeability to FITC-dextran 70 in monolayers of cells expressing wild-type and cytosolic eNOS. PAF failed to increase permeability to FITC-dextran 70 in monolayers of cells transfected with eNOS targeted to the plasma membrane. Interestingly, this occurred despite eNOS Ser-1177 phosphorylation and production of comparable amounts of NO. Our results demonstrate that the presence of eNOS in the cytosol is necessary for PAF-induced hyperpermeability. Our data provide new insights into the dynamics of eNOS and eNOS-derived NO in the process of inflammation.
authors.affiliation: Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07101-1709; Instituto de Inmunología, Escuela de Medicina, Universidad Austral de Chile, Valdivia 511-0566, Chile.
authors.forename: Fabiola A
authors.initials: FA
authors.lastname: Sánchez
authors.affiliation: Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07101-1709.
authors.forename: Roshniben
authors.initials: R
authors.lastname: Rana
authors.affiliation: Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07101-1709.
authors.forename: Francisco G
authors.initials: FG
authors.lastname: González
authors.affiliation: Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07101-1709.
authors.forename: Toru
authors.initials: T
authors.lastname: Iwahashi
authors.affiliation: Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07101-1709.
authors.forename: Ricardo G
authors.initials: RG
authors.lastname: Durán
authors.affiliation: Department of Pharmacology and Toxicology, Georgia Health Sciences University-Medical College of Georgia, Augusta, Georgia 30912.
authors.forename: David J
authors.initials: DJ
authors.lastname: Fulton
authors.affiliation: Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07101-1709.
authors.forename: Annie V
authors.initials: AV
authors.lastname: Beuve
authors.affiliation: Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07101-1709.
authors.forename: David D
authors.initials: DD
authors.lastname: Kim
authors.affiliation: Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07101-1709. Electronic address: duran@umdnj.edu.
authors.forename: Walter N
authors.initials: WN
authors.lastname: Durán
mesh_terms: D002138:Calibration; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D007249:Inflammation; D008856:Microscopy, Fluorescence / Q000379:methods; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry / Q000502:physiology*; D010539:Permeability; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000378:metabolism; D021381:Protein Transport; D013347:Subcellular Fractions
chemical_list: D018076:DNA, Complementary; D010972:Platelet Activating Factor; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III