Peroxynitrite causes endothelial cell monolayer barrier dysfunction

Nitric oxide (·NO) attenuates hydrogen peroxide (H₂O₂)-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 (O₂^-) 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 μM) or 3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 μM). 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 β-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 β-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.