Increased Nitric Oxide Synthase Activity as a Cause of Mitochondrial Dysfunction in Rat Hepatocytes: Roles for Tumor Necrosis Factor a

vestigate whether Kupffer cell–derived mediators alter Kupffer cells have been identified as the first and most the mitochondrial oxidative phosphorylation of hepatoavid scavenger cells for lipopolysaccharide (LPS), the endocytes in the endotoxemic condition. Liver cells were isotoxins of gram-negative intestinal bacteria, and a potent imlated from male Wistar rats. Oxidative phosphorylation munomodulator passing into the liver through the portal was monitored as the fluorescence of rhodamine 123 vein. Phagocytosis, i.e., the uptake of particles, by macro(Rh123), which is the fluorescent cationic dye used to phages usually leads to the release of specific molecules. LPS indicate mitochondrial energy synthesis. Two hours triggers the production of immunomodulating and inflammaafter coculture of hepatocytes with lipopolysaccharide tory mediators such as cytokines, lipid mediators, and active (LPS)-pretreated Kupffer cells, a marked decrease in heoxygen species from Kupffer cells, which are recognized in patocyte rhodamine 123 fluorescence was observed. The playing a role in the pathogenesis of liver injury. hepatocyte mitochondrial dysfunction was attenuated A specific, inorganic signal molecule that a variety of cells by the addition of either N-monomethyl-L-arginine (Lsynthesize and excrete is nitric oxide (NO). Although NO NMMA), an inhibitor of nitric oxide (NO) synthesis, or was first identified as an endothelium-derived relaxing factor aminoguanidine, an inducible-type of NO synthase invia the stimulation of soluble guanylate cyclase, it appears hibitor, to the culture medium of cocultures, to the preto cause intracellular iron loss, to modulate mitochondrial treatment of LPS-activated Kupffer cells with antisense respiration, to inactivate aconitase and ribonucleotide reducoligodeoxynucleotides against iNOS messenger RNA tase, to inhibit DNA synthesis, and to promote cytostasis in (mRNA), or to tumor necrosis factor a (TNF-a) mRNA. target cells. Kupffer cells have been shown to synthesize Four hours after the coculture, hepatocyte Rh123 fluoand to release NO in response to cytokines, interferon rescence further decreased, and an iNOS induction as gamma, and LPS. There is a growing body of evidence that well as an increased NO production were observed in indicates that an excessive release of NO leads to the inhibihepatocytes that were cocultured with LPS-pretreated tion of hepatic mitochondrial metabolisms that are characterKupffer cells. The membrane barrier dysfunction of heized by diminished oxidative phosphorylation and that play patocytes, indicated by propidium iodide staining, was a role in the process of hepatic damage. However, there have also induced by a 4-hour coculture with LPS-pretreated been few investigations using an in vitro coculture system. Kupffer cells. These late-phase changes were inhibited Cellular adenine triphosphate depletion is a common feature either by the pretreatment of hepatocytes with antiof hypoxic and toxic injury. In hepatocellular injury that is sense oligodeoxynucleotides against iNOS mRNA or by induced by various oxidant chemicals, the disruption of mitotreatments that are effective in the early phase (within chondrial adenine triphosphate formation appears to be a 2 hours). Incubation with recombinant rat TNF-a decommon mechanism leading to lethal cell death. However, creased hepatocyte Rh123 fluorescence within 2 hours. the regulatory mechanisms of NO-mediated hepatocyte inThus, the present study suggests that NO and TNF-a jury, i.e., the cell source of NO, are still under debate. released from LPS-pretreated Kupffer cells directly inRecent studies have indicated that hepatocytes themselves hibit the hepatocyte mitochondrial function in the early are the major source of NO, because the combination of some phase, and then NO synthesized by TNF-a–induced hecytokines stimulates the iNOS (inducible nitric oxide synpatocyte iNOS causes lethal hepatocyte injury, characthase) induction of hepatocytes. Curran et al. have reterized by diminished mitochondrial energization and ported that inflammatory mediators that are released from