Nitric oxide mediates Kupffer cell-induced reduction of mitochondrial energization in hepatoma cells: a comparison with oxidative burst.
The metabolic changes in rat hepatoma cell line, AH70 cells, after coculturing with Kupffer cells were visualized using a silicon-intensified target camera and subsequent processing with a computer-assisted digital imaging processor. In cocultured tumor cells, nonactivated Kupffer cells reduced mitochondrial energization as indicated by the decrease in the fluorescence intensity of rhodamine 123 (Rh123) and induced lipid peroxidation as shown by the dichlorofluorescein (DCF) activation. The reduction in Rh123 could be eliminated by addition of an analogue of L-arginine (NG-monomethyl-L-arginine), suggesting the involvement of nitric oxide (NO.) in the decrease in mitochondrial energization. Superoxide dismutase did not inhibit the reduction in Rh123 but significantly inhibited DCF activation. These findings indicate that the latter reaction was mediated by superoxide anion. Two h after the cells were cocultured, propidium iodide-positive, severely injured tumor cells significantly increased in number. This increase was significantly attenuated by addition of NG-monomethyl-L-arginine but not by superoxide dismutase, suggesting that NO. may be greatly involved in Kupffer cell-mediated injury of AH70 cells. In another set of experiments, the culture medium of Kupffer cells caused no significant alteration of Rh123, DCF, and propidium iodide-associated fluorescences in AH70 cells. In addition, ultrastructural observation revealed that the membrane-to-membrane attachment between Kupffer cells and tumor cells occurred within 30 min after coculturing. These results suggest that Kupffer cell-derived NO. release, triggered by the close contact with tumor cells, may induce damage to tumor cells via inhibition of mitochondrial energization.
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
I
I
Kurose
S
S
Miura
D
D
Fukumura
Y
Y
Yonei
H
H
Saito
S
S
Tada
M
M
Suematsu
M
M
Tsuchiya
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002450:Cell Communication; D002452:Cell Count; D005456:Fluorescent Dyes / Q000378:metabolism; D007728:Kupffer Cells / Q000502:physiology*; D015227:Lipid Peroxidation; D008113:Liver Neoplasms / Q000378:metabolism*; D008297:Male; D008930:Mitochondria, Liver / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D020112:Rhodamine 123; D012235:Rhodamines / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D012235:Rhodamines; D020112:Rhodamine 123; D009569:Nitric Oxide; D013482:Superoxide Dismutase