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

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 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.

Fig. 1.

Functional Characterization of ECV-CD8-GFPeNOSmyr⁻. (A) PAF significantly increases phosphorylation of eNOS at Ser-1177 as a function of time (n = 3). (B) PAF induces eNOS dephosphorylation at Thr-495 (n = 3). (C) PAF stimulates a robust increase in pericellular NO concentration (mean ± SEM; n = 6; *, P < 0.05). (D) 10⁻⁷ M PAF increases permeability to FITC-Dx-70 in CD8-GFPeNOSmyr⁻ cells. Data are mean ± SEM. (*, P < 0.05, n = 4. (E) PAF induces eNOS translocation in CD8-GFPeNOSmyr⁻ cells. The Western blotting shows that PAF induces the disappearance of eNOS from plasma membrane (n = 3).

Fig. 2.

Inhibition of caveolar internalization decreases PAF-induced hyperpermeability in ECV-CD8-GFPeNOSmyr⁻ cells. (A) Western blotting showing that cotransfection of dyn2K44A inhibits PAF-induced eNOS endocytosis. (B) Western blotting showing that cotransfection of cav1Y14F inhibits PAF-induced eNOS endocytosis. (C) Transfection of ECV-CD8-GFPeNOSmyr⁻ cells with dyn2K44A inhibits PAF-induced hyperpermeability to FITC-DX-70 (*, P < 0.05 vs. control, n = 5). (D) Transfection of ECV-CD8-GFPeNOSmyr⁻ cells with cav1Y14F inhibits PAF-induced hyperpermeability to FITC-DX-70. (*, P < 0.05 vs. control, n = 5).

Fig. 3.

Inhibition of eNOS endocytosis blocks hyperpermeability independent of cell type. Different treatments that prevent caveolar endocytosis of eNOS in different cells block PAF-induced hyperpermeability. (A) Transfection of CVEC with cav1Y14F. (B) Treatment of CVEC with 5 mM cyclodextrin (CD). (C) Treatment of ECVeNOSGFP cells with 10 mM cyclodextrin.

Fig. 4.

Effect of inhibition of PAF-induced eNOS endocytosis in ECV-CD8-GFPeNOSmyr⁻ cells and CVEC. Except for control, all measurements were performed after stimulation with 10⁻⁷ M PAF. (A–D) The times on the bar graphs are in the same order as in the Western blotting panels. (A) Phosphorylation of CD8-GFPeNOSmyr⁻ in the presence of the dynamin mutant. (B) Phosphorylation of eNOS in CVEC in the presence of the dynamin mutant. (C) Phosphorylation of CD8-GFPeNOSmyr⁻ in the presence of the caveolin mutant. (D) Phosphorylation of eNOS in CVEC in the presence of the caveolin mutant. (E) NO production in CVEC transfected with dyn2K44A, cav1Y14F, or treated with 5 mM cyclodextrin (Cyd).

Fig. 5.

Inhibition of permeability and eNOS translocation by cav1Y14F. (A) Inhibition of eNOS endocytosis abolishes VEGF-induced hyperpermeability. Transfection of CVEC with cav1Y14F significantly inhibited VEGF-induced hyperpermeability to FITC-DX-70 (*, P < 0.05 vs. control, n = 5). (B) Transfection of CVEC with cav1Y14F blocks eNOS translocation to cytosol. PAF and VEGF decrease the brightness of eNOS (red fluorescence) from the plasma membrane relative to control. Transfection with cav1Y14F inhibits the PAF- and VEGF-induced movement of eNOS away from the plasma membrane.