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

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 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.

Fig. 1.

Platelet-activating factor (PAF; 100 nM) induces endothelial nitric oxide (NO) synthase (eNOS) phosphorylation and NO production in postcapillary venular endothelial cells (CVECs). A: immunofluorescence shows eNOS expression in cell membrane and Golgi in control CVECs. B: PAF significantly stimulated a robust increase in pericellular NO concentration. The induced response was rapid, peaked at about 3 min, and returned to basal levels ∼10 min after PAF application (means ± SE, n = 6 experiments, P < 0.05). C: PAF significantly increased phosphorylation of eNOS at Ser¹¹⁷⁷ and decreased eNOS phosphorylation at Thr⁴⁹⁵ as a function of time, as indicated by the ratio of phosphorylated (p-eNOS) to total eNOS (*P < 0.05, n = 3 experiments). The Western blots illustrate typical examples of changes in eNOS phosphorylation at Ser¹¹⁷⁷ and Thr⁴⁹⁵.

Fig. 2.

Depletion of eNOS inhibits PAF-induced hyperpermeability. A: 100 nM PAF increases permeability to FITC-dextran 70 KDa (Dx-70) in CVECs. Data are expressed as permeability coefficients (means ± SE). *P < 0.05, n = 5 experiments. B: Western blots show depletion of eNOS as a function of small interfering (si)RNA concentration and time. C: depletion of endogenous eNOS in CVECs abrogates the development of PAF-induced hyperpermeability. CVECs transfected with scrambled (sc) siRNA served as a control. The increase in permeability elicited by 100 nM PAF is significant compared with all other interventions (means ± SE; *P < 0.05, n = 5 experiments).

Fig. 3.

PAF induces eNOS translocation in CVECs. A: immunofluorescence images of CVECs were obtained in control cells and after 100 nM PAF treatment. The image shows that PAF induces the disappearance of eNOS from plasma membrane and its appearance in a diffuse fashion in cytosol. The images are representative of 3 independent experiments. B: Western blots of isolated lipid rafts in control and PAF-treated cells (fraction 1, lightest; fraction 12, heaviest). Fractions were probed against eNOS and caveolin.

Fig. 4.

Inhibition of caveolar internalization decreases PAF-induced hyperpermeability in CVECs. A: expression of dyn2K44A in transfected CVECs. B: impact of CVEC transfection with dyn2K44A on permeability to FITC-DX-70. CVECs transfected with the corresponding empty vector served as control. PAF induced a robust hyperpermeability in the control CVECs. Transfection of CVEC monolayers with dyn2K44A significantly inhibited the PAF-induced hyperpermeability to FITC-DX-70 (*P < 0.05 compared with control and interventions, n = 5 experiments).

Fig. 5.

Dyn2K44A inhibits eNOS traffic in CVECs. eNOS inmunofluorescence images taken from CVEC monolayers at the end of the measurement of permeability. In the control (baseline) CVEC, eNOS is distributed in the cell membrane and in the Golgi area. Nontransfected CVECs show eNOS translocation to cytosol (diffuse material) after challenge with PAF (middle). In contrast, eNOS is efficaciously retained in the plasma membrane in the dyn2K44A-transfected CVECs after administration of 100 nM PAF.