Lipopolysaccharide and interferon-gamma-induced nitric oxide production and protein oxidation in mouse peritoneal macrophages are affected by glutathione peroxidase-1 gene knockout.
This study investigated the role of glutathione peroxidase-1 (GPX1) in protein oxidation in peritoneal macrophages. Macrophages isolated from both wild-type (WT) and GPX1 knockout (KO) mice were activated by lipopolysaccharide (LPS, 1 microg/ml) and interferon-gamma (IFN, 10 U/ml for 24 or 48 h in the presence or absence of 1 microM diquat (DQ), 250 microM aminoguanidine (AG, an inhibitor of inducible nitric oxide synthase), and (or) 100 microM diethyldithiocarbamate (DETC, an inhibitor of Cu,Zn-SOD). In the KO macrophages, there was no protein band detected by Western blot with anti-GPX1 antibody and 98% reduction in total GPX activity compared with WT cells. Nitric oxide (NO) synthesis was greatly enhanced after 24 h by GPX1 knockout and DQ, but inhibited by AG or DETC. Protein carbonyl formation in total cell extract was clearly associated with NO synthesis as higher levels of protein carbonyl were detected in activated KO than WT macrophages, and DQ enhanced slightly while AG or DETC virtually blocked its formation. A similarly marginal effect of GPX1 KO was observed on protein nitration. The LPS/IFN/DQ-induced DNA fragmentation was blocked by AG, but not by DETC. Cell viability at 48 h was decreased by the LPS/IFN activation and further reduced by the addition of DQ, but restored by AG. In conclusion, GPX1 affects the NO production in activated peritoneal macrophages and protects these cells against NO-associated protein oxidation.
Fu
Y
Y
Department of Animal Science, Cornell University, Ithaca, NY 14853, USA.
McCormick
C C
CC
Roneker
C
C
Lei
X G
XG
D000818:Animals; D015153:Blotting, Western; D002470:Cell Survival; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D004050:Ditiocarb / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000502:physiology*; D006146:Guanidines / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000187:drug effects* / Q000201:enzymology; D051379:Mice; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009573:Nitrites / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D000094765:Glutathione Peroxidase GPX1
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005609:Free Radicals; D006146:Guanidines; D008070:Lipopolysaccharides; D009573:Nitrites; D012333:RNA, Messenger; D009569:Nitric Oxide; D007371:Interferon-gamma; D004050:Ditiocarb; D005979:Glutathione Peroxidase; D019001:Nitric Oxide Synthase; D013482:Superoxide Dismutase; C004479:pimagedine; D000094765:Glutathione Peroxidase GPX1; C000722068:Gpx1 protein, mouse