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LPS‐Induced Hyper‐permeability in Human Lung Microvascular Endothelial Cells (HLMVEC) Involves the Nitration‐Mediated Activation of RhoA
Author(s) -
Rafikov Ruslan,
Aggarwal Saurabh,
Hou Yali,
Snead Connie,
Catravas John,
Black Stephen
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.1101.2
Subject(s) - rhoa , nitration , permeability (electromagnetism) , chemistry , vascular permeability , microbiology and biotechnology , lung , biophysics , cancer research , medicine , pathology , biochemistry , biology , signal transduction , organic chemistry , membrane
Acute lung injury (ALI) is characterized by endothelial hyper‐permeability and activation of the small GTPase, RhoA. The mechanisms underlying the activation of RhoA are unresolved. Increased protein nitration correlates with lung endothelial hyper‐permeability in the LPS exposed mouse and the present study was designed to investigate the potential of nitration events to modulate RhoA activity. LPS induced both the nitration and activation of RhoA in HLMVEC. Mass spectroscopy localized the nitration site to Y34 and a decoy peptide containing this sequence prevented the LPS‐mediated nitration and activation of RhoA. In additional experiments, HLMVEC monolayers were grown on specially constructed wells to measure transendothelial resistance (TER). The LPS mediated reduction in TER was attenuated by the Y34 decoy peptide but not by a peptide in which the Y34 was replaced by phenylalanine (Y34F). Utilizing recombinant human RhoA protein along with mant‐GDP and mant‐GTP fluorescence and stop‐flow kinetics, we found that peroxynitrite decreased the binding reaction constant of mant‐GDP, but not mant‐GTP, to RhoA suggesting that nitrated RhoA binds GDP less tightly, stimulating GDP‐GTP turnover increasing RhoA activity. We conclude that the enhanced RhoA activation during ALI is due, at least in part, to the nitration of RhoA and agents that prevent this event could have clinical utility in the management of ALI.