During endotoxemia, hepatic endothelial cells can be ex-The study aimed to assess the effect of lipopolysacchaposed to oxidative stress, because sinusoidal endothelial cells ride (LPS) in vivo (from Escherichia coli, 2 mg/kg body are the first sites of neutrophil adhesion and are in direct weight intraperitoneally) on the production and elimicontact with activated Kupffer cells. 1 Studies have indicated nation of hydrogen peroxide (H 2 O 2 ) in rat hepatic endothat the injury of sinusoidal microvasculature is one of the thelial and Kupffer cells. Twenty-two hours after the inearliest events in the sequel of developing hepatic failure jection of LPS, hepatic cells were isolated by collagenase during severe sepsis, endotoxemia, or reperfusion injury. 2-4 and pronase digestion followed by centrifugal elutria-
The cellular events that protect against exogenous oxidative tion, and cell-associated H 2 O 2 was determined by flow stress are not well understood in hepatic cells. Studies demcytometry analysis using 2,7-dichloroflorescin diaceonstrate that during endotoxemia [5][6][7] or following cytokine 8 tate (DCF-diacetate). LPS treatment did not alter the and phagocytic challenges, 9 hepatic macrophages and sinubasal or phorbol myristate acetate-stimulated levels of soidal endothelial cells display high rates of glucose uptake H 2 O 2 -related fluorescence in endothelial cells; however, and catabolism in the hexose monophosphate shunt (HMS). it doubled phorbol myristate acetate-stimulated fluo-Glucose catabolism in the HMS generates reduced nicotinrescence in Kupffer cells. Administration of varying conamide-adenine dinucleotide phosphate (NADPH), which is centrations of H 2 O 2 (range, 10 07 -10 04 mol/L) in vitro the primary reducing equivalent for the synthesis of superoxcaused a significantly delayed increase in fluorescence ide anion or nitric oxide by NADPH oxidase and nitric oxide in endothelial cells from endotoxemic rats as compared synthetase, respectively. 10,11 Following the dismutation of with cells from saline-injected animals. The 50% effective these compounds, the elimination of hydrogen peroxide concentration of H 2 O 2 was found at 1.1 1 10 06 and 8.1
or peroxinitrite is also dependent on NADPH, because 1 10 06 mol/L on endothelial cells after saline and LPS this reduced coenzyme is required for the functions of glutatreatment, respectively. No differences were detected in thione peroxidase (via glutathione) and for the stability of H 2 O 2 -stimulated fluorescence between resting and LPScatalase. 12,13 Therefore, the metabolism of glucose through stimulated Kupffer cells. Administration of varying gluthe HMS is potentially important for either the production cose concentrations in vitro significantly decreased the or the elimination of reactive oxygen species (ROS) in cells H 2 O 2 -stimulated fluorescence in endothelial and Kupffer dependent on the use of exogenous glucose. We showed precells from LPS-injected animals. Inhibition of nitric oxviously that the addition of lipopolysaccharide (LPS) in vivo ide synthase by in vitro administration of N G -monoresulted in elevated gene expression of glucose transporter methyl-L-arginine (L-NMMA) did not alter the H 2 O 2 -or GLUT1, glucose-6-phosphate dehydrogenase (the rate-limphorbol myristate acetate-stimulated responses in eniting enzyme of the HMS), and Mn-dependent superoxide dothelial and Kupffer cells. As shown earlier, LPS stimudismutase in both endothelial and Kupffer cells. 14,15 However, lates the gene expression of GLUT1 glucose transporter, gene expression of Se-dependent glutathione peroxidase and glucose-6-phosphate dehydrogenase (G6PD), superoxide CuZn-dependent superoxide dismutase was elevated in endodismutases, and glutathione peroxidase in hepatic endothelial cells only. 15 The LPS-induced responses of these functhelial cells. The present data indicate that the LPS-intionally related genes of glucose metabolism and ROS-elimiduced metabolic alterations are accompanied by an nating pathways suggested that activated endothelial cells increased H 2 O 2 -detoxifying capacity in hepatic endothemay have an increased capacity to metabolize hydrogen perlial cells. This may represent a protective mechanism oxide (H 2 O 2 ). Therefore, in the present study, we aimed to against exogenous oxidative stress caused by activated determine whether endotoxemia alters H 2 O 2 detoxifying achepatic phagocytes during inflammation. Our observativity in freshly isolated hepatic endothelial and Kupffer tions are consistent with primed production of reactive cells. We investigated H 2 O 2 metabolism 22 hours after a sinoxygen species (ROS) in LPS-activated Kupffer cells. gle LPS injection intraperitoneally, because this treatment (HEPATOLOGY 1996;24:691-696.) caused alterations in the gene expression of glucose metabolic enzymes and ROS-eliminating pathways in a cell-specific fashion. 15 We also tested the effect of glucose availability and the potential involvement of nitric oxide on H 2 O 2 metabolism Abbreviations: HMS, hexose monophosphate shunt; NADPH, reduced nicotinamide ade-in these cells.
nine dinucleotide phosphate; ROS, reactive oxygen species; LPS, lipopolysaccharide; DCFdiacetate, 2,7-dicholorfluorescin diacetate; L-NMMA, N G -monomethyl-L-arginine; PMA,
Methods
phorbol 12-myristate 13-acetate.