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This Article Full Text Full Text (PDF) All Versions of this Article: 295/4/H1642    most recent 00629.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Sánchez, F. A. Articles by Durán, W. N. Search for Related Content PubMed PubMed Citation Articles by Sánchez, F. A. Articles by Durán, W. N.

Internalization of eNOS via caveolae regulates PAF-induced inflammatory hyperpermeability to macromolecules

¹Program in Vascular Biology, Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey; and ²Department of Systems Biology and Translational Medicine, Texas A&M Health Science Center, Temple, Texas

Submitted 15 June 2008 ; accepted in final form 12 August 2008

Endothelial nitric oxide (NO) synthase (eNOS) is thought to regulate microvascular permeability via NO production. We tested the hypotheses that the expression of eNOS and eNOS endocytosis by caveolae are fundamental for appropriate signaling mechanisms in inflammatory endothelial permeability to macromolecules. We used bovine coronary postcapillary venular endothelial cells (CVECs) because these cells are derived from the microvascular segment responsible for the transport of macromolecules in inflammation. We stimulated CVECs with platelet-activating factor (PAF) at 100 nM and measured eNOS phosphorylation, NO production, and CVEC monolayer permeability to FITC-dextran 70 KDa (Dx-70). PAF translocated eNOS from plasma membrane to cytosol, induced changes in the phosphorylation state of the enzyme, and increased NO production from 4.3 ± 3.8 to 467 ± 22.6 nM. PAF elevated CVEC monolayer permeability to FITC-Dx-70 from 3.4 ± 0.3 x 10^–6 to 8.5 ± 0.4 x 10^–6 cm/s. The depletion of endogenous eNOS with small interfering RNA abolished PAF-induced hyperpermeability, demonstrating that the expression of eNOS is required for inflammatory hyperpermeability responses. The inhibition of the caveolar internalization by blocking caveolar scission using transfection of dynamin dominant-negative mutant, dyn2K44A, inhibited PAF-induced hyperpermeability to FITC-Dx-70. We interpret these data as evidence that 1) eNOS is required for hyperpermeability to macromolecules and 2) the internalization of eNOS via caveolae is an important mechanism in the regulation of endothelial permeability. We advance the novel concept that eNOS internalization to cytosol is a signaling mechanism for the onset of microvascular hyperpermeability in inflammation.

endothelial nitric oxide; endothelial cells; microvascular permeability; endothelial nitric oxide synthase translocation; acetylcholine; platelet-activating factor

This article has been cited by other articles:

				F. A. Sanchez, R. Rana, D. D. Kim, T. Iwahashi, R. Zheng, B. K. Lal, D. M. Gordon, C. J. Meininger, and W. N. Duran Internalization of eNOS and NO delivery to subcellular targets determine agonist-induced hyperpermeability PNAS, April 21, 2009; 106(16): 6849 - 6853. [Abstract] [Full Text] [PDF]