Direct evidence that induced nitric oxide production in hepatocytes prevents liver damage during lipopolysaccharide tolerance in rats.
BACKGROUND
The role of nitric oxide (NO) in lipopolysaccharide (LPS) tolerance in the liver has been investigated in a number of previous studies, but it is still not clear whether NO is cytotoxic or cytoprotective. The aims of this study were to investigate whether low-dose LPS (LLPS)-induced hepatic production of NO is beneficial and to clarify the origins of cytoprotective NO-producing cells in the liver during LPS tolerance.


METHODS
Male Wistar rats received saline or LLPS intraperitoneally (i.p.; 0.01-1000 microg/kg) followed by a high dose of LPS (HLPS, 5 mg/kg) at various time intervals (4-16 h). NG-nitro-L-arginine methyl ester (L-NAME) was used to investigate the effects of inhibition of NOS. 4,5-Diaminofluorescein (DAF-2) was used to identify NO-producing cells in isolated liver cells in vitro. At various time points (4-16 h) after saline or LLPS (1 microg/kg, i.p.) injection, hepatocytes and Kupffer cells were isolated, incubated in 7 microm DAF-2 diacetate, and perfused with Krebs solution. Illumination at 495 nm revealed DAF-fluorescence (515 nm) in isolated cells under confocal laser fluorescence microscopy. The NO production in hepatocytes and Kupffer cells was assessed by the number of labeled cells per 1000 cells or per 100 cells, respectively.


RESULTS
Pretreatment with LLPS (0.1-100 microg/kg) resulted in a significant reduction (maximal at 8 h) of the HLPS-induced liver damage. L-NAME abolished the LLPS-induced protection. The NO production in hepatocytes was significantly increased and reached a maximum of 84% of all cells 8 h after LLPS administration. By contrast, the NO production in Kupffer cells remained constant at 95%, even following preinjection of LLPS.


CONCLUSIONS
LLPS-induced NO in hepatocytes, but not in Kupffer cells, exhibits cytoprotective effects on HLPS-induced liver damage, suggesting that NO has a beneficial role in the induction of the early phase of LPS tolerance.
Ebisawa
Yoshiaki
Y
Department of Surgery II, Asahikawa Medical College, Asahikawa, Hokkaido, Japan.
Kono
Toru
T
Yoneda
Masashi
M
Asama
Toshiyuki
T
Chisato
Naoyuki
N
Sugawara
Mutsubu
M
Ishikawa
Kazushi
K
Iwamoto
Jun
J
Ayabe
Tokiyoshi
T
Kohgo
Yutaka
Y
Kasai
Shinichi
S
D000818:Animals; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019793:Fluorescein; D007202:Indicators and Reagents; D007728:Kupffer Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008107:Liver Diseases / Q000378:metabolism* / Q000517:prevention & control*; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
C111205:4,5-diaminofluorescein; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D008070:Lipopolysaccharides; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D019793:Fluorescein; D019331:NG-Nitroarginine Methyl Ester