Premium
eNOS Internalization Mediated by Caveolar Endocytosis Regulates Microvascular Permeability.
Author(s) -
Sanchez Fabiola Alejandra,
Kim David D,
Duran Ricardo G,
Meininger Cynthia J,
Duran Walter N
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.926.10
Subject(s) - internalization , enos , endocytosis , caveolae , microbiology and biotechnology , dynamin , chemistry , nitric oxide , biology , nitric oxide synthase , biochemistry , signal transduction , cell , organic chemistry
Endothelial nitric oxide synthase (eNOS) is highly regulated through phosphorylation, protein interactions and subcellular location. We demonstrated that platelet‐activating factor (PAF) activates eNOS and induces enzyme internalization in association with hyperpermeability. Here, we tested two hypotheses A) eNOS endocytosis regulates endothelial hyperpermeability to macromolecules, and B) eNOS internalization is mediated by caveolar endocytosis. We used bovine coronary postcapillary venular endothelial cells (CVEC). PAF increased NO production from 4.3 ± 3.8 to 467 ± 22.6 nmol/L and permeability to FITC‐dextran‐70 from 2.0 ± 0.23 × 10 −6 cm/s to 6.0 ± 1.0 × 10 −6 cm/s. PAF translocated eNOS from plasma membrane to cytosol. Application of 5 mM cyclodextrin (CD) and transfection of CVEC with dyn2K44A (a dynamin dominant negative mutant) blocked NO production and inhibited permeability responses to PAF. We used biotinylation of ECV304‐CD8eNOS‐GFP cells and Western blotting to show that eNOS is internalized via caveolae in response to PAF. Our data demonstrate that eNOS is internalized via caveolar endocytosis, and that this process is involved regulating endothelial permeability in response to inflammatory agonists. (Supported by NIH grant 5RO1 HL706434‐05).