Internalization of eNOS via caveolae regulates PAF-induced inflammatory hyperpermeability to macromolecules.
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.
Sánchez
Fabiola A
FA
Program in Vascular Biology, Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey, USA. sanchefa@umdnj.edu
Kim
David D
DD
Durán
Ricardo G
RG
Meininger
Cynthia J
CJ
Durán
Walter N
WN
D000818:Animals; D002199:Capillary Permeability; D002417:Cattle; D021941:Caveolae / Q000201:enzymology*; D002478:Cells, Cultured; D003911:Dextrans / Q000378:metabolism; D034285:Dynamin II / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis; D042783:Endothelial Cells / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D007249:Inflammation / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D003911:Dextrans; D010972:Platelet Activating Factor; D034741:RNA, Small Interfering; C015219:fluorescein isothiocyanate dextran; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D034285:Dynamin II; D016650:Fluorescein-5-isothiocyanate