Peroxynitrite causes endothelial cell monolayer barrier dysfunction

James L. Knepler, Loui N. Taher, Mahesh P. Gupta, Carolyn Patterson, Fred Pavalko, Michael D. Ober, C. Michael Hart

Research output: Contribution to journalArticlepeer-review

79 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)C1064-C1075
JournalAmerican Journal of Physiology - Cell Physiology
Issue number3 50-3
StatePublished - 2001


  • Actin
  • Catenin
  • Nitrotyrosine

ASJC Scopus subject areas

  • Physiology
  • Cell Biology


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