Internalization of eNOS and NO delivery to subcellular targets determine agonist-induced hyperpermeability

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 106; no. 16; pp. 6849 - 6853
• Main Authors: Sánchez, Fabiola A, Rana, Roshniben, Kim, David D, Iwahashi, Toru, Zheng, Ruifang, Lal, Brajesh K, Gordon, Donna M, Meininger, Cynthia J, Durán, Walter N
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 21.04.2009; National Acad Sciences
• Subjects: Animals; Biological Sciences; Capillary Permeability - drug effects; Cattle; Caveolae; Caveolae - drug effects; Caveolae - metabolism; Cell membranes; Complex Systems: From Chemistry to Systems Biology Special Feature; Cyclodextrins; Endocytosis; Endocytosis - drug effects; Endothelial cells; Endothelial Cells - cytology; Endothelial Cells - drug effects; Endothelial Cells - enzymology; Enzyme Activation - drug effects; Internalization; Membranes; Molecular biology; Mutation; Nitric oxide; Nitric Oxide - metabolism; Nitric Oxide Synthase Type III - metabolism; Oxides; Phosphorylation; Physiological regulation; Plasma; Platelet Activating Factor - pharmacology; Recombinant Fusion Proteins - metabolism; Subcellular Fractions - drug effects; Subcellular Fractions - enzymology; Transfection; Vascular Endothelial Growth Factor A - pharmacology
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0812694106

The molecular mechanisms of endothelial nitric oxide synthase (eNOS) regulation of microvascular permeability remain unresolved. Agonist-induced internalization may have a role in this process. We demonstrate here that internalization of eNOS is required to deliver NO to subcellular locations to increase endothelial monolayer permeability to macromolecules. Using dominant-negative mutants of dynamin-2 (dyn2K44A) and caveolin-1 (cav1Y14F), we show that anchoring eNOS-containing caveolae to plasma membrane inhibits hyperpermeability induced by platelet-activating factor (PAF), VEGF in ECV-CD8eNOSGFP (ECV-304 transfected cells) and postcapillary venular endothelial cells (CVEC). We also observed that anchoring caveolar eNOS to the plasma membrane uncouples eNOS phosphorylation at Ser-1177 from NO production. This dissociation occurred in a mutant- and cell-dependent way. PAF induced Ser-1177-eNOS phosphorylation in ECV-CD8eNOSGFP and CVEC transfected with dyn2K44A, but it dephosphorylated eNOS at Ser-1177 in CVEC transfected with cav1Y14F. Interestingly, dyn2K44A eliminated NO production, whereas cav1Y14F caused reduction in NO production in CVEC. NO production by cav1Y14F-transfected CVEC occurred in caveolae bound to the plasma membrane, and was ineffective in causing an increase in permeability. Our study demonstrates that eNOS internalization is required for agonist-induced hyperpermeability, and suggests that a mechanism by which eNOS is activated by phosphorylation at the plasma membrane and its endocytosis is required to deliver NO to subcellular targets to cause hyperpermeability.