Peroxynitrite causes endothelial cell monolayer barrier dysfunction.
Nitric oxide (.NO) attenuates hydrogen peroxide (H(2)O(2))-mediated barrier dysfunction in cultured porcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD, Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am J Physiol Lung Cell Mol Physiol 280: L116-L126, 2001). However,.NO rapidly combines with superoxide (O) to form the powerful oxidant peroxynitrite (ONOO(-)), which we hypothesized would cause PAEC monolayer barrier dysfunction. To test this hypothesis, we treated PAEC with ONOO(-) (500 microM) or 3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 microM). SIN-1-mediated ONOO(-) formation was confirmed by monitoring the oxidation of dihydrorhodamine 123 to rhodamine. Both ONOO(-) and SIN-1 increased albumin clearance (P < 0.05) in the absence of cytotoxicity and altered the architecture of the cytoskeletal proteins actin and beta-catenin as detected by immunofluorescent confocal imaging. ONOO(-)-induced barrier dysfunction was partially reversible and was attenuated by cysteine. Both ONOO(-) and SIN-1 nitrated tyrosine residues, including those on beta-catenin and actin, and oxidized proteins in PAEC. The introduction of actin treated with ONOO(-) into PAEC monolayers via liposomes also resulted in barrier dysfunction. These results indicate that ONOO(-) directly alters endothelial cytoskeletal proteins, leading to barrier dysfunction.
Knepler
J L
JL
Department of Medicine, Richard L. Roudebush Veterans Affairs and Indiana University Medical Centers, Indianapolis, Indiana 46202, USA.
Taher
L N
LN
Gupta
M P
MP
Patterson
C
C
Pavalko
F
F
Ober
M D
MD
Hart
C M
CM
D000199:Actins / Q000378:metabolism; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D007700:Kinetics; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology*; D009566:Nitrates / Q000494:pharmacology*; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D011651:Pulmonary Artery; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000494:pharmacology; D013552:Swine; D015534:Trans-Activators; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D051176:beta Catenin
D000199:Actins; D003598:Cytoskeletal Proteins; D009566:Nitrates; D020030:Nitric Oxide Donors; D016877:Oxidants; D017382:Reactive Oxygen Species; D015534:Trans-Activators; D051176:beta Catenin; C039642:peroxynitric acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; C002385:linsidomine; D008981:Molsidomine; D013482:Superoxide Dismutase