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.