AMP-activated protein kinase deficiency reduces ozone-induced lung injury and oxidative stress in miceReport as inadecuate




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Respiratory Research

, 12:64

First Online: 01 December 2011Received: 13 January 2011Accepted: 19 May 2011

Abstract

BackgroundAcute ozone exposure causes lung oxidative stress and inflammation leading to lung injury. At least one mechanism underlying the lung toxicity of ozone involves excessive production of reactive oxygen and nitrogen intermediates such as peroxynitrite. In addition and beyond its major prooxidant properties, peroxynitrite may nitrate tyrosine residues altering phosphorylation of many protein kinases involved in cell signalling. It was recently proposed that peroxynitrite activates 5-AMP-activated kinase AMPK, which regulates metabolic pathways and the response to cell stress. AMPK activation as a consequence of ozone exposure has not been previously evaluated. First, we tested whether acute ozone exposure in mice would impair alveolar fluid clearance, increase lung tissue peroxynitrite production and activate AMPK. Second, we tested whether loss of AMP-activated protein kinase alpha1 subunit in mouse would prevent enhanced oxidative stress and lung injury induced by ozone exposure.

MethodsControl and AMPKα1 deficient mice were exposed to ozone at a concentration of 2.0 ppm for 3 h in glass cages. Evaluation was performed 24 h after ozone exposure. Alveolar fluid clearance AFC was evaluated using fluorescein isothiocyanate tagged albumin. Differential cell counts, total protein levels, cytokine concentrations, myeloperoxidase activity and markers of oxidative stress, i.e. malondialdehyde and peroxynitrite, were determined in bronchoalveolar lavage BAL and lung homogenates LH. Levels of AMPK-Thr phosphorylation and basolateral membrane Na+-K+-ATPase abundance were determined by Western blot.

ResultsIn control mice, ozone exposure induced lung inflammation as evidence by increased leukocyte count, protein concentration in BAL and myeloperoxidase activity, pro-inflammatory cytokine levels in LH. Increases in peroxynitrite levels 3 vs 4.4 nM, p = 0.02 and malondialdehyde concentrations 110 vs 230 μmole-g wet tissue were detected in LH obtained from ozone-exposed control mice. Ozone exposure consistently increased phosphorylated AMPK-Thr to total AMPK ratio by 80% in control mice. Ozone exposure causes increases in AFC and basolateral membrane Na+-K+-ATPase abundance in control mice which did not occur in AMPKα1 deficient mice.

ConclusionsOur results collectively suggest that AMPK activation participates in ozone-induced increases in AFC, inflammation and oxidative stress. Further studies are needed to understand how the AMPK pathway may provide a novel approach for the prevention of ozone-induced lung injury.

AbbreviationsAFCAlveolar fluid clearance

AMPK5-AMP-activated protein kinase

BALbronchoalveolar lavage

DHR123dihydrorhodamine-123

G-CSFgranulocyte colonystimulating factor

GLUTglucose transporter

GM-CSFgranulocyte-macrophage colony stimulating factor

IFNinterferon

ILinterleukin

MCPmonocyte chemotactic protein

MDAmalondialdehyde

MIPmacrophage inflammatory protein

MPOmyeloperoxidase

NADPH oxidasenicotinamide adenine dinucleotide phosphate oxidase

PKCprotein kinase C

PMNpolymorphonuclear leukocyte

ppmparts-million

RANTESRegulated on Activation Normal T Cell Expressed and Secreted

SDS-PAGESodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

TNFtumor necrosis factor

Electronic supplementary materialThe online version of this article doi:10.1186-1465-9921-12-64 contains supplementary material, which is available to authorized users.

Sébastien Hulo, Hélène Tiesset, Steve Lancel, Jean J Louis Edmé , Benoit Viollet, Annie Sobaszek and Rémi Nevière contributed equally to this work.

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Author: Sébastien Hulo - Hélène Tiesset - Steve Lancel - Jean J Louis Edmé - Benoit Viollet - Annie Sobaszek - Rémi Nevière

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