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.

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

“…5B shows a series of images demonstrating that: (i) PAF and VEGF induce translocation of eNOS from the plasma membrane (caveolae) to the cytosol in postcapillary ECs; and (ii) transfection of ECs with cav1Y14F inhibits the translocation of eNOS. Translocation of eNOS is indicated mainly by the disappearance of eNOS from the cell plasma membrane and the enhanced brightness in the cytosol (6,7).…”

“…We previously showed that eNOS internalization (endocytosis) is a required step in the signaling cascade leading to PAF-induced hyperpermeability (7). In this study, we tested the hypothesis that caveolar internalization of eNOS is a requirement for localized NO production and NO delivery to a subcellular target to cause hyperpermeability.…”

“…Agonists such as platelet-activating factor (PAF) and VEGF phosphorylate eNOS via Akt (8, 9). Despite advances in our understanding of the biochemistry of eNOS, the mechanisms by which these molecular modifications determine the functional outcome of eNOS activation remain unexplored.NO derived from eNOS is a key mediator in the hyperpermeability response to PAF and VEGF (7,10,11). We previously showed that eNOS internalization (endocytosis) is a required step in the signaling cascade leading to PAF-induced hyperpermeability (7).…”

“…NO derived from eNOS is a key mediator in the hyperpermeability response to PAF and VEGF (7,10,11). We previously showed that eNOS internalization (endocytosis) is a required step in the signaling cascade leading to PAF-induced hyperpermeability (7).…”

“…The enzyme is modified by N-myristoylation and palmitoylation, which targets it to the caveolae in the plasma membrane where it is kept in a basal state by binding to caveolin-1 through a consensus site (1,2). Agonists activate eNOS through multiple mechanisms: phosphorylation/ dephosphorylation of specific residues, interaction with different proteins, S-nitrosylation, and specific subcellular localization (1,(3)(4)(5)(6)(7). Agonists such as platelet-activating factor (PAF) and VEGF phosphorylate eNOS via Akt (8,9).…”