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

Fabiola A. Sánchez, David D. Kim, Ricardo G. Durán, Cynthia J. Meininger, Walter N. Durán

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

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 FITCdextran 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 FITCDx-70 from 3.4 ± 0.3 × 10-6 to 8.5 ± 0.4 × 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.

Original languageEnglish (US)
Pages (from-to)H1642-H1648
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume295
Issue number4
DOIs
StatePublished - Oct 1 2008
Externally publishedYes

Fingerprint

Caveolae
Platelet Activating Factor
Permeability
Endothelial Cells
Nitric Oxide
Cytosol
Phosphorylation
Inflammation
Dynamins
Fluorescein-5-isothiocyanate
Nitric Oxide Synthase Type III
Capillary Permeability
Endocytosis
Small Interfering RNA
Transfection
Cell Membrane
Enzymes

All Science Journal Classification (ASJC) codes

  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)
  • Physiology

Keywords

  • Acetylcholine
  • Endothelial cells
  • Endothelial nitric oxide
  • Endothelial nitric oxide synthase translocation
  • Microvascular permeability
  • Platelet-activating factor

Cite this

Sánchez, Fabiola A. ; Kim, David D. ; Durán, Ricardo G. ; Meininger, Cynthia J. ; Durán, Walter N. / Internalization of eNOS via caveolae regulates PAF-induced inflammatory hyperpermeability to macromolecules. In: American Journal of Physiology - Heart and Circulatory Physiology. 2008 ; Vol. 295, No. 4. pp. H1642-H1648.
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Internalization of eNOS via caveolae regulates PAF-induced inflammatory hyperpermeability to macromolecules. / Sánchez, Fabiola A.; Kim, David D.; Durán, Ricardo G.; Meininger, Cynthia J.; Durán, Walter N.

In: American Journal of Physiology - Heart and Circulatory Physiology, Vol. 295, No. 4, 01.10.2008, p. H1642-H1648.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Internalization of eNOS via caveolae regulates PAF-induced inflammatory hyperpermeability to macromolecules

AU - Sánchez, Fabiola A.

AU - Kim, David D.

AU - Durán, Ricardo G.

AU - Meininger, Cynthia J.

AU - Durán, Walter N.

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Y1 - 2008/10/1

N2 - 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 FITCdextran 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 FITCDx-70 from 3.4 ± 0.3 × 10-6 to 8.5 ± 0.4 × 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.

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KW - Acetylcholine

KW - Endothelial cells

KW - Endothelial nitric oxide

KW - Endothelial nitric oxide synthase translocation

KW - Microvascular permeability

KW - Platelet-activating factor

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