INCREASED NITRIC-OXIDE SYNTHASE ACTIVITY AS A CAUSE OF MITOCHONDRIAL DYSFUNCTION IN RAT HEPATOCYTES - ROLES FOR TUMOR-NECROSIS-FACTOR-ALPHA

Kupffer cells have been implicated in playing an important role in the pathogenesis of endotoxemia-associated liver injury. The present stud y was designed to investigate whether Kupffer cell-derived mediators a lter the mitochondrial oxidative phosphorylation of hepatocytes in the endotoxemic condition. Liver cells were isolated from male Wistar rat s. Oxidative phosphorylation was monitored as the fluorescence of rhod amine 123 (Rh123), which is the fluorescent cationic dye used to indic ate mitochondrial energy synthesis. Two hours after coculture of hepat ocytes with lipopolysaccharide (LPS)-pretreated Kupffer cells, a marke d decrease in hepatocyte rhodnmine 123 fluorescence was observed. The hepatocyte mitochondrial dysfunction was attenuated by the addition of either N-G-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxi de (NO) synthesis, or aminoguanidine, an inducible-type of NO synthase inhibitor, to the culture medium of cocultures, to the pretreatment o f LPS-activated Kupffer cells with antisense oligodeoxynucleotides aga inst iNOS messenger RNA (mRNA), or to tumor necrosis factor alpha (TNF -alpha) mRNA. Four hours after the coculture, hepatocyte Rh123 fluores cence further decreased, and an iNOS induction as well as an increased NO production were observed in hepatocytes that were cocultured with LPS-pretreated Kupffer cells. The membrane barrier dysfunction of hepa tocytes, indicated by propidium iodide staining, was also induced by a 4-hour coculture with LPS-pretreated Kupffer cells. These late-phase changes were inhibited either by the pretreatment of hepatocytes with antisense oligodeoxynucleotides against iNOS mRNA or by treatments tha t are effective in the early phase (within 2 hours). Incubation with r ecombinant rat TNF-alpha decreased hepatocyte Rh123 fluorescence withi n 2 hours. Thus, the present study suggests that NO and TNF-alpha rele ased from LPS-pretreated Kupffer cells directly inhibit the hepatocyte mitochondrial function in the early phase, and then NO synthesized by TNF-alpha-induced hepatocyte iNOS causes lethal hepatocyte injury, ch aracterized by diminished mitochondrial energization and membrane barr ier function in the late phase.