Nitric oxide mediates mitochondrial dysfunction in hepatoma cells induced by non-activated Kupffer cells: evidence implicating ICAM-1-dependent process.
The metabolic changes in a rat hepatoma cell line, AH70 cells, after co-culture with rat Kupffer cells (KC) were visualized and analysed using a fluorescence microscope equipped with a silicon intensified target camera and a laser scanning confocal microscopic system. Kupffer cells were isolated from male Wistar rats, and cultured without any stimuli. The non-activated KC reduced the mitochondrial energization in the cocultured AH70 cells within 2 h, which was indicated by decreased rhodamine 123 (Rh123) fluorescence. Either NG-monomethyl-L-arginine or dexamethasone significantly attenuated the KC-induced mitochondrial dysfunction in AH70 cells, suggesting the involvement of nitric oxide (NO) derived from inducible-type nitric oxide synthase (iNOS). Administration of monoclonal antibody (mAb) directed against rat ICAM-1 also prevented the decrease in Rh123 fluorescence. Electron microscopy revealed that the membrane-to-membrane attachment between KC and AH70 cells occurred within 2 h. A laser scanning confocal microscopic observation using mAb against ICAM-1 presented that the ICAM-1 expression on AH70 cells and KC increased after the co-culture. It is therefore concluded that the KC-mediated mitochondrial dysfunction of hepatoma cells largely depends on NO production by iNOS. Furthermore, the present study supports a scenario that the NO production and release from KC is triggered by the close contact with hepatoma cells through adhesion molecules such as ICAM-1.
Kurose
I
I
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Ebinuma
H
H
Higuchi
H
H
Yonei
Y
Y
Saito
H
H
Kato
S
S
Miura
S
S
Ishii
H
H
D000818:Animals; D000911:Antibodies, Monoclonal; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D002448:Cell Adhesion; D018920:Coculture Techniques; D003907:Dexamethasone / Q000494:pharmacology; D005456:Fluorescent Dyes; D018799:Intercellular Adhesion Molecule-1 / Q000502:physiology*; D007728:Kupffer Cells / Q000502:physiology*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008297:Male; D008856:Microscopy, Fluorescence; D008930:Mitochondria, Liver / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D051381:Rats; D017208:Rats, Wistar; D020112:Rhodamine 123; D012235:Rhodamines; D014407:Tumor Cells, Cultured; D019323:omega-N-Methylarginine
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D012235:Rhodamines; D018799:Intercellular Adhesion Molecule-1; D020112:Rhodamine 123; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D003907:Dexamethasone; D001120:Arginine; D019001:Nitric Oxide Synthase