Role in nitric oxide in Kupffer cell-mediated hepatoma cell cytotoxicity in vitro and ex vivo.
The metabolic change in tumor cells (AH70, a rat hepatoma cell line) cocultured with isolated rat Kupffer cells were visualized and analyzed by a laser scanning confocal imaging system. When AH70 cells were cocultured with Kupffer cells, fluorescence intensity of rhodamine 123 (Rh123) decreased, indicating the reduction of mitochondrial function. The reduction in Rh123 was eliminated by NG-monomethyl-L-arginine (L-NMMA), an analogue of L-arginine, suggesting the involvement of nitric oxide (NO). Two hour after the cells were cocultured, membrane compromised AH70 cells which were observed as propidium from 2.8% to 25%. This increase was also attenuated by L-NMMA, suggesting that Kupffer cell-mediated injury of tumor cells largely depends on NO. The concentrations of NO-2 + NO-3 in the culture medium markedly increased after coculture of AH70 cells with Kupffer cells. Moreover, NO synthase (NOS) activity in Kupffer cells significantly increased after coculture. These in vitro results suggest that NO mediates Kupffer cell-induced tumor cell damage characterized by reduced mitochondrial function and diminished barrier function. In the ex vivo study of the perfused liver to which AH70 cells were injected via the catheter inserted into the portal vein, some AH70 cells were arrested in the upper stream of sinusoid and the fluorescence intensity of Rh123 in adherent AH70 cells decreased in a time-dependent manner within 2 hours. The number of PI-positive AH70 cells also increased 2 hours after the injection of AH70 cells. These changes were inhibited by either administration of N omega-L-nitroarginine-methylester (L-NAME) to perfusate or pretreatment of the rat liver with GdCl3, which is known to deplete Kupffer cell function. Thus, the present study suggests that NO from Kupffer cells induces mitochondrial dysfunction in tumor cells followed by membrane barrier dysfunction in the liver sinusoid.
Fukumura
D
D
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Yonei
Y
Y
Kurose
I
I
Saito
H
H
Ohishi
T
T
Higuchi
H
H
Miura
S
S
Kato
S
S
Kimura
H
H
Ebinuma
H
H
Ishi
H
H
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D007700:Kinetics; D007728:Kupffer Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D008114:Liver Neoplasms, Experimental / Q000473:pathology*; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D020112:Rhodamine 123; D012235:Rhodamines; D014407:Tumor Cells, Cultured; D019323:omega-N-Methylarginine
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007527:Isoenzymes; D012235:Rhodamines; D020112:Rhodamine 123; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase