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Department of Anatomy, Cell Biology, and Injury Sciences, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103
The present study investigated the effect of lipopolysaccharide (LPS; from Escherichia coli, 2 mg/kg body wt ip) on selected aspects of the antioxidant status in Kupffer and sinusoidal endothelial cells. Cells were isolated 18 h after the injection of saline or LPS. In fresh suspension cultures, cellular reduced glutathione (GSH) and H2O2 were determined by monochlorobimane, and 2',7'-dichlorofluorescein diacetate, respectively, using a fluorescence plate reader. LPS injection increased GSH content two- to threefold in Kupffer cells compared with cells from control rats. Cellular GSH content was higher in endothelial than Kupffer cells. However, LPS did not increase GSH content in endothelial cells. Addition of H2O2 (40-200 µM) to Kupffer or endothelial cells caused a transient decrease in GSH, which was more pronounced in cells from control rats (~45% drop) than in LPS-exposed cells (~25% drop). Depleted GSH levels were accompanied by a proportional increase in cellular H2O2. After inhibition of catalase by 3-amino-1,2,4-triazole, the presence of 0.2 mM H2O2 depleted GSH content by 75% and 40% in Kupffer cells from saline- or LPS-injected rats, respectively. The same treatments caused a similar 50% decrease in both activated and control endothelial cells. LPS decreased catalase activity by 45% in Kupffer cells, whereas it had no effect on catalase in endothelial cells. Glutathione reductase activity was not altered by LPS in either cell type. These data show that in activated Kupffer cells the elevated level of cellular glutathione plays an augmented role in the protection against reactive oxygen species, whereas the contribution of catalase to H2O2 detoxification is attenuated. In LPS-stimulated endothelial and Kupffer cells, the efficient maintenance of GSH is consistent with upregulated production of reducing power through the hexose phosphate shunt observed previously.
aminotriazol; antioxidants; glutathione reductase; endothelium; glucose-6-phosphate dehydrogenase; glutathione peroxidase; hepatocytes; hexose monophosphate shunt; inflammation; liver; lipopolysaccharide; macrophages; reactive oxygen species; superoxide anion
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