Effect of antioxidants on lipopolysaccharide-stimulated induction of mangano superoxide dismutase mRNA in bovine pulmonary artery endothelial cells.
Generation of reactive oxygen species (ROS) is a common event in the pathogenesis of acute lung injury. Endothelial cells may be both a target and a source of the ROS. Exposure of bovine pulmonary endothelial cells (BPAEC) to lipopolysaccharide (LPS) has been shown to result in intracellular generation of both ROS and the antioxidant enzyme, mangano superoxide dismutase (MnSOD). The present study investigates whether alterations in intracellular oxidant state affect LPS-stimulated cytotoxicity and induction of MnSOD mRNA. BPAEC were pretreated with either the free radical scavenger, dimethylsulfoxide (DMSO), the xanthine oxidase inhibitor, allopurinol, or N-acetylcysteine (a cysteine derivate capable of increasing glutathione stores) prior to exposure to LPS (0.1 microgram/ml) for either 4, 8 or 18 hours. We found that pretreatment of BPAEC with DMSO blocked both LPS-induced cytotoxicity and induction of the MnSOD gene. Nuclear run-off experiments demonstrated that LPS-stimulated induction of the MnSOD mRNA occurred at the transcriptional level and that DMSO blocked this event. Pretreatment with allopurinol also prevented the cytotoxicity associated with LPS but, in contrast to DMSO, did not alter induction of MnSOD mRNA. N-acetylcysteine did not affect the LPS-stimulated cytotoxicity but resulted in an early and transient reduction in induction of the MnSOD gene. We conclude that LPS stimulates generation of intracellular ROS that regulate induction of the MnSOD gene at the transcriptional level further, we conclude that LPS-stimulated cytotoxicity involves both the xanthine oxidase pathway and perhaps intracellular generation of hydroxyl radicals. The difference in the protective effect between DMSO, NAC and allopurinol suggest that upregulation of the MnSOD gene does not contribute to LPS-induced cytotoxicity.
Department of Medicine, Vanderbilt University Medical Center, Nasville, Tennessee 37232-2650, USA.
J
J
Mitchell
H
H
Jiang
L
L
Berry
B
B
Meyrick
D000111:Acetylcysteine / Q000494:pharmacology; D000493:Allopurinol / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D015152:Blotting, Northern; D002417:Cattle; D002467:Cell Nucleus; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004727:Endothelium / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008853:Microscopy; D011651:Pulmonary Artery; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*
D000975:Antioxidants; D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; D000493:Allopurinol; D013482:Superoxide Dismutase; D000111:Acetylcysteine; D004121:Dimethyl Sulfoxide