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Internalization of eNOS and NO Delivery to Subcellular Targets Determine Agonist-Induced Hyperpermeability

Fabiola A. Sánchez, Roshniben Rana, David D. Kim, Toru Iwahashi, Ruifang Zheng, Brajesh K. Lal, Donna M. Gordon, Cynthia J. Meininger, Walter N. Durán and Shu Chien
Proceedings of the National Academy of Sciences of the United States of America
Vol. 106, No. 16 (Apr. 21, 2009), pp. 6849-6853
Stable URL: http://www.jstor.org/stable/40482185
Page Count: 5
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Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
Internalization of eNOS and NO Delivery to Subcellular Targets Determine Agonist-Induced Hyperpermeability
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Abstract

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 plateletactivating 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 ECVCD8eNOSGFP 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.

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