Up-regulation of the peroxiredoxin-6 related metabolism of reactive oxygen species in skeletal muscle of mice lacking neuronal nitric oxide synthase.
Although neuronal nitric oxide synthase (nNOS) plays a substantial role in skeletal muscle physiology, nNOS-knockout mice manifest an only mild phenotypic malfunction in this tissue. To identify proteins that might be involved in adaptive responses in skeletal muscle of knockout mice lacking nNOS, 2D-PAGE with silver-staining and subsequent tandem mass spectrometry (LC-MS/MS) was performed using extracts of extensor digitorum longus muscle (EDL) derived from nNOS-knockout mice in comparison to C57Bl/6 control mice. Six proteins were significantly (P < or = 0.05) more highly expressed in EDL of nNOS-knockout mice than in that of C57 control mice, all of which are involved in the metabolism of reactive oxygen species (ROS). These included prohibitin (2.0-fold increase), peroxiredoxin-3 (1.9-fold increase), Cu(2+)/Zn(2+)-dependent superoxide dismutase (SOD; 1.9-fold increase), heat shock protein beta-1 (HSP25; 1.7-fold increase) and nucleoside diphosphate kinase B (2.6-fold increase). A significantly higher expression (4.1-fold increase) and a pI shift from 6.5 to 5.9 of peroxiredoxin-6 in the EDL of nNOS-knockout mice were confirmed by quantitative immunoblotting. The concentrations of the mRNA encoding five of these proteins (the exception being prohibitin) were likewise significantly (P < or = 0.05) higher in the EDL of nNOS-knockout mice. A higher intrinsic hydrogen peroxidase activity (P < or = 0.05) was demonstrated in EDL of nNOS-knockout mice than C57 control mice, which was related to the presence of peroxiredoxin-6. The treatment of mice with the chemical NOS inhibitor L-NAME for 3 days induced a significant 3.4-fold up-regulation of peroxiredoxin-6 in the EDL of C57 control mice (P < or = 0.05), but did not alter its expression in EDL of nNOS-knockout mice. ESR spectrometry demonstrated the levels of superoxide to be 2.5-times higher (P < or = 0.05) in EDL of nNOS-knockout mice than in C57 control mice while an in vitro assay based on the emission of 2,7-dichlorofluorescein fluorescence disclosed the concentration of ROS to be similar in both strains of mice. We suggest that the up-regulation of proteins that are implicated in the metabolism of ROS, particularly of peroxiredoxin-6, within skeletal muscles of nNOS-knockout mice functionally compensates for the absence of nNOS in scavenging of superoxide.
Da Silva-Azevedo
Luis
L
Department of Physiology, Charité-Campus Benjamin Franklin, Arnimallee 22, Berlin-Dahlem, Germany.
Jähne
Sebastian
S
Hoffmann
Christian
C
Stalder
Daniel
D
Heller
Manfred
M
Pries
Axel R
AR
Zakrzewicz
Andreas
A
Baum
Oliver
O
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008401:Gas Chromatography-Mass Spectrometry; D055551:HSP27 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D054778:NM23 Nucleoside Diphosphate Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000172:deficiency* / Q000235:genetics; D054465:Peroxiredoxin VI / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000091402:Prohibitins; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D015854:Up-Regulation
D004791:Enzyme Inhibitors; D055551:HSP27 Heat-Shock Proteins; D054778:NM23 Nucleoside Diphosphate Kinases; D000091402:Prohibitins; D012097:Repressor Proteins; D006861:Hydrogen Peroxide; D054465:Peroxiredoxin VI; D052248:Nitric Oxide Synthase Type I; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; C517941:Nme2 protein, mouse; D019331:NG-Nitroarginine Methyl Ester