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eNOS Internalization Mediated by Caveolar Endocytosis Regulates Microvascular Permeability
Author(s) -
Sanchez Fabiola A,
Kim David D,
Durán Ricardo G,
Lal Brajesh K,
Durán Walter N
Publication year - 2007
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.21.5.a490-b
Subject(s) - endocytosis , internalization , enos , microbiology and biotechnology , caveolae , chemistry , nitric oxide , cell , nitric oxide synthase , biology , biochemistry , signal transduction , organic chemistry
NO is an important regulator of endothelial function. Endothelial nitric oxide synthase (eNOS) is highly regulated through phosphorylation, protein interactions and subcellular location. We demonstrated that eNOS activation mediated by PAF (platelet‐activating factor) induces enzyme internalization (AJP 291 :, 2006) in association with hyperpermeability. We tested two related hypothesis eNOS internalization is mediated by caveolar endocytosis, and endocytosis regulates increases in endothelial permeability to macromolecules. We used ECV304 cells permanently transfected with CD8eNOSGFPmyr‐ and PAF as the agonist. PAF increases permeability to FITC‐dextran 70 from 8.2×10 −7 ±3.9×10 −7 to 1.0×10 −5 ±9.7×10 −7 cm/s in ECV‐CD8eNOSGFPmyr‐ cells. Treatment with 5 mM cyclodextrin (CD) inhibits the response to PAF. We used biotin labeling of cell surface proteins, followed by neutravidin precipitation and western blot for eNOS, to test for the movement of CD8eNOSGFPmyr‐. PAF induced internalization of CD8eNOSGFPmyr‐, as indicated by a 20% reduction in biotinylated‐cell surface eNOS. We conclude that PAF induces translocation of eNOS by caveolar endocytosis, and that eNOS endocytosis is required for PAF‐stimulated hyperpermeability to macromolecules. (Supported by NIH grant 5RO1 HL706434‐05).