ABSTRACT
The enzyme xanthine oxidase (XO) has been implicated in the pathogenesis of several disease processes, such as ischemia-reperfusion injury, because of its ability to generate reactive oxygen species. The expression of XO and its precursor xanthine dehydrogenase (XDH) is regulated at pre- and posttranslational levels by agents such as lipopolysaccharide and hypoxia. Posttranslational modification of the protein, for example through thiol oxidation or proteolysis, has been shown to be important in converting XDH to XO. The possibility of posttranslational modification of XDH/XO through phosphorylation has not been adequately investigated in mammalian cells, and studies have reported conflicting results. The present report demonstrates that XDH/XO is phosphorylated in rat pulmonary microvascular endothelial cells (RPMEC) and that phosphorylation is greatly increased ( approximately 50-fold) in response to acute hypoxia (4 h). XDH/XO phosphorylation appears to be mediated, at least in part, by casein kinase II and p38 kinase as inhibitors of these kinases partially prevent XDH/XO phosphorylation. In addition, the results indicate that p38 kinase, a stress-activated kinase, becomes activated in response to hypoxia (an approximately 4-fold increase after 1 h of exposure of RPMEC to hypoxia) further supporting a role for this kinase in hypoxia-stimulated XDH/XO phosphorylation. Finally, hypoxia-induced XDH/XO phosphorylation is accompanied by a 2-fold increase in XDH/XO activity, which is prevented by inhibitors of phosphorylation. In summary, this study shows that XDH/XO is phosphorylated in hypoxic RPMEC through a mechanism involving p38 kinase and casein kinase II and that phosphorylation is necessary for hypoxia-induced enzymatic activation.
Subject(s)
Hypoxia , Xanthine Dehydrogenase/metabolism , Xanthine Oxidase/metabolism , Animals , Arsenites/pharmacology , Blotting, Western , Casein Kinase II , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Endothelium, Vascular/metabolism , Enzyme Activation , Enzyme Inhibitors/pharmacology , Indicators and Reagents/pharmacology , Lung/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation/drug effects , Precipitin Tests , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/metabolism , Rats , Signal Transduction , Sodium Compounds/pharmacology , Sorbitol/pharmacology , Xanthine Dehydrogenase/chemistry , Xanthine Oxidase/chemistry , p38 Mitogen-Activated Protein KinasesABSTRACT
Early excision and graft surgery is now a routinely used procedure, but whereas its application for burns of the hand has been well determined it is employed less extensively for facial lesions. It is a heavy and very hemorrhagic surgery requiring close cooperation between surgeons and intensive care physicians. Its use for the face is not easy to define, difficulties arising because of the need to establish the depth of the burn, the hemorrhagic risk involved and the need for effective compression after graft surgery.
