title: Hyper-activation of pp60Srclimits nitric oxide signaling by increasing asymmetric dimethylarginine levels during acute lung injury.
abstract: The molecular mechanisms by which the endothelial barrier becomes compromised during lipopolysaccharide (LPS) mediated acute lung injury (ALI) are still unresolved. We have previously reported that the disruption of the endothelial barrier is due, at least in part, to the uncoupling of endothelial nitric oxide synthase (eNOS) and increased peroxynitrite-mediated nitration of RhoA. The purpose of this study was to elucidate the molecular mechanisms by which LPS induces eNOS uncoupling during ALI. Exposure of pulmonary endothelial cells (PAEC) to LPS increased pp60Srcactivity and this correlated with an increase in nitric oxide (NO) production, but also an increase in NOS derived superoxide, peroxynitrite formation and 3-nitrotyrosine (3-NT) levels. These effects could be simulated by the over-expression of a constitutively active pp60Src(Y527FSrc) mutant and attenuated by over-expression of dominant negative pp60Srcmutant or reducing pp60Srcexpression. LPS induces both RhoA nitration and endothelial barrier disruption and these events were attenuated when pp60Srcexpression was reduced. Endothelial NOS uncoupling correlated with an increase in the levels of asymmetric dimethylarginine (ADMA) in both LPS exposed and Y527FSrc over-expressing PAEC. The effects in PAEC were also recapitulated when we transiently over-expressed Y527FSrc in the mouse lung. Finally, we found that the pp60-Src-mediated decrease in DDAH activity was mediated by the phosphorylation of DDAH II at Y207 and that a Y207F mutant DDAH II was resistant to pp60Src-mediated inhibition. We conclude that pp60Srccan directly inhibit DDAH II and this is involved in the increased ADMA levels that enhance eNOS uncoupling during the development of ALI.
authors.affiliation: Vascular Biology Center and the Center for Biotechnology & Genomic Medicine, Augusta University, Augusta, GA, United States.
authors.forename: Sanjiv
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Department of Medicine, The University of Arizona, Tucson, AZ, United States.
authors.forename: Xutong
authors.initials: X
authors.lastname: Sun
authors.affiliation: Department of Medicine, The University of Arizona, Tucson, AZ, United States.
authors.forename: Satish Kumar
authors.initials: SK
authors.lastname: Noonepalle
authors.affiliation: Department of Medicine, The University of Arizona, Tucson, AZ, United States.
authors.forename: Qing
authors.initials: Q
authors.lastname: Lu
authors.affiliation: Department of Medicine, The University of Arizona, Tucson, AZ, United States.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Zemskov
authors.affiliation: Department of Medicine, The University of Arizona, Tucson, AZ, United States.
authors.forename: Ting
authors.initials: T
authors.lastname: Wang
authors.affiliation: Department of Anesthesiology, The University of Alabama, Birmingham, AL, United States.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Aggarwal
authors.affiliation: Vascular Biology Center and the Center for Biotechnology & Genomic Medicine, Augusta University, Augusta, GA, United States.
authors.forename: Christine
authors.initials: C
authors.lastname: Gross
authors.affiliation: Center for Biotechnology & Genomic Medicine, Old Dominion University, Norfolk, VA, United States.
authors.forename: Shruti
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Department of Medicine, The University of Arizona, Tucson, AZ, United States.
authors.forename: Ankit A
authors.initials: AA
authors.lastname: Desai
authors.affiliation: Vascular Biology Center and the Center for Biotechnology & Genomic Medicine, Augusta University, Augusta, GA, United States.
authors.forename: Yali
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Vascular Biology Center and the Center for Biotechnology & Genomic Medicine, Augusta University, Augusta, GA, United States.
authors.forename: Sridevi
authors.initials: S
authors.lastname: Dasarathy
authors.affiliation: Department of Medicine, The University of Arizona, Tucson, AZ, United States.
authors.forename: Ning
authors.initials: N
authors.lastname: Qu
authors.affiliation: Vascular Biology Center and the Center for Biotechnology & Genomic Medicine, Augusta University, Augusta, GA, United States.
authors.forename: Vijay
authors.initials: V
authors.lastname: Reddy
authors.affiliation: Vascular Biology Center and the Center for Biotechnology & Genomic Medicine, Augusta University, Augusta, GA, United States.
authors.forename: Sung Gon
authors.initials: SG
authors.lastname: Lee
authors.affiliation: Vascular Biology Center and the Center for Biotechnology & Genomic Medicine, Augusta University, Augusta, GA, United States.
authors.forename: Mary
authors.initials: M
authors.lastname: Cherian-Shaw
authors.affiliation: Department of Medicine, The University of Arizona, Tucson, AZ, United States.
authors.forename: Jason X-J
authors.initials: JX
authors.lastname: Yuan
authors.affiliation: Center for Biotechnology & Genomic Medicine, Old Dominion University, Norfolk, VA, United States.
authors.forename: John D
authors.initials: JD
authors.lastname: Catravas
authors.affiliation: Department of Medicine, The University of Arizona, Tucson, AZ, United States.
authors.forename: Ruslan
authors.initials: R
authors.lastname: Rafikov
authors.affiliation: Department of Medicine, The University of Arizona, Tucson, AZ, United States.
authors.forename: Joe G N
authors.initials: JGN
authors.lastname: Garcia
authors.affiliation: Department of Medicine, The University of Arizona, Tucson, AZ, United States. Electronic address: steveblack@email.arizona.edu.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Black
mesh_terms: D055371:Acute Lung Injury / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation; D008070:Lipopolysaccharides / Q000633:toxicity; D051379:Mice; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics* / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000096:biosynthesis; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics* / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; C487735:dimethylarginine; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D001120:Arginine; D052250:Nitric Oxide Synthase Type III; D016392:Proto-Oncogene Proteins pp60(c-src); D000581:Amidohydrolases; C059999:dimethylargininase
keywords: Acute lung injury; Signal transduction; eNOS uncoupling