Increased nitric oxide synthase activity as a cause of mitochondrial dysfunction in rat hepatocytes: Roles for tumor necrosis factor α

Iwao Kurose, Soichiro Miura, Hajime Higuchi, Naoyuki Watanabe, Yoshitaka Kamegaya, Masaaki Takaishi, Kengo Tomita, Dai Fukumura, Shinzo Kato, Hiromasa Ishii

Research output: Contribution to journalArticlepeer-review

47 Citations (Scopus)


Kupffer cells have been implicated in playing an important role in the pathogenesis of endotoxemia-associated liver injury. The present study was designed to investigate whether Kupffer cell-derived mediators alter the mitochondrial oxidative phosphorylation of hepatocytes in the endotoxemic condition. Liver cells were isolated from male Wistar rats. Oxidative phosphorylation was monitored as the fluorescence of rhodamine 123 (Rh123), which is the fluorescent cationic dye used to indicate mitochondrial energy synthesis. Two hours after coculture of hepatocytes with lipopolysaccharide (LPS)-pretreated Kupffer cells, a marked decrease in hepatocyte rhodamine 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 oxide (NO) synthesis, or aminoguanidine, an inducible- type of NO synthase inhibitor, to the culture medium of cocultures, to the pre-treatment of LPS-activated Kupffer cells with antisense oligodeoxynucleotides against iNOS messenger RNA (mRNA), or to tumor necrosis factor α (TNF-α) mRNA. Four hours after the coculture, hepatocyte Rh123 fluorescence 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 hepatocytes, 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 that are effective in the early phase (within 2 hours). Incubation with recombinant rat TNF-α decreased hepatocyte Rh123 fluorescence within 2 hours. Thus, the present study suggests that NO and TNF-α released from LPS-pretreated Kupffer cells directly inhibit the hepatocyte mitochondrial function in the early phase, and then NO synthesized by TNF-α-induced hepatocyte iNOS causes lethal hepatocyte injury, characterized by diminished mitochondrial energization and membrane barrier function in the phase.

Original languageEnglish
Pages (from-to)1185-1192
Number of pages8
Issue number5
Publication statusPublished - 1996 Nov
Externally publishedYes

ASJC Scopus subject areas

  • Hepatology


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