Peroxynitrite causes endothelial cell monolayer barrier dysfunction

Nitric oxide (. NO) attenuates hydrogen peroxide (H2O2)-mediated barrier dy sfunction in cultured porcine pulmonary artery endothelial cells (PAEC) (Gu pta MP, Ober MD,. Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am J Physiol Lung Cell Mol Physiol 280: L116-LI26, 2001). However, . NO rapidl y combines with superoxide (O-2(-)) to form the powerful oxidant peroxynitr ite (ONOO-), which we hypothesized would cause PAEC monolayer barrier dysfu nction. To test this hypothesis, we treated PAEC with ONOO- (500 muM) or 3- morpholinosydnonimine hydrochloride (SIN-1; 1-500 muM). SIN-1-mediated ONOO - formation was confirmed by monitoring the oxidation of dihydrorhodamine 1 23 to rhodamine. Both ONOO- and SIN-1 increased albumin clearance (P < 0.05 ) in the absence of cytotoxicity and altered the architecture of the cytosk eletal proteins actin and <beta>-catenin as detected by immunofluorescent c onfocal imaging. ONOO--induced barrier dysfunction was partially reversible and was attenuated by cysteine. Both ONOO- and SIN-1 nitrated tyrosine res idues, including those on beta -catenin and actin, and oxidized proteins in PAEC. The introduction of actin treated with ONOO- into PAEC monolayers vi a liposomes also resulted in barrier dysfunction. These results indicate th at ONOO- directly alters endothelial cytoskeletal proteins, leading to barr ier dysfunction.