Kurose I, Kato S, Ishii H, Fukumura D, Miura S, Suematsu M, Tsuchiya M
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Hepatology. 1993 Aug;18(2):380-8.
The influence of endogenous nitric oxide, which is generated by stimulation with lipopolysaccharide, on the mitochondrial energization of rat hepatocytes was investigated in vitro and ex vivo. Using a fluorescence microscope equipped with a silicon intensifier target camera, we visualized fluorescence of rhodamine-123, a mitochondrial energization-sensitive fluorescence probe, in individual hepatocytes and measured the fluorescence intensity with a digital imaging processor. Although addition of Kupffer cells or lipopolysaccharide in a range of 0.1 to 1.0 microgram/ml caused no significant alteration in the fluorescence in hepatocytes, Kupffer cells plus 1.0 microgram/ml lipopolysaccharide reduced fluorescence intensity in the cocultured hepatocytes. The alteration of rhodamine-123 fluorescence in the hepatocytes induced by lipopolysaccharide-activated Kupffer cells was significantly inhibited by the addition of NG-monomethyl-L-arginine, a selective inhibitor of nitric oxide synthesis. The transportal infusion of lipopolysaccharide also decreased rhodamine-123 fluorescence in perfused rat liver. The decrease was significantly enhanced in the pericentral regions. Autofluorescence of NADH was elicited by continuous infusion of lipopolysaccharide; this reaction was also enhanced in the pericentral regions. We showed the main site of uptake of infused lipopolysaccharide in the hepatic lobule to be in the periportal regions with fluorescein isothiocyanate-labeled lipopolysaccharide. Our results indicate that the inhibition of mitochondrial energization occurs mainly in pericentral regions, which are distant from the lipopolysaccharide uptake site. The continuous administration of NG-monomethyl-L-arginine significantly attenuated the lipopolysaccharide-induced decrease in rhodamine-123 fluorescence and increase of the NADH contents of the hepatic lobule.(ABSTRACT TRUNCATED AT 250 WORDS)
在体外和体内研究了脂多糖刺激产生的内源性一氧化氮对大鼠肝细胞线粒体能量代谢的影响。使用配备硅增强靶相机的荧光显微镜，我们观察了单个肝细胞中线粒体能量代谢敏感荧光探针罗丹明-123的荧光，并使用数字成像处理器测量了荧光强度。尽管添加库普弗细胞或0.1至1.0微克/毫升范围内的脂多糖不会导致肝细胞荧光发生显著变化，但库普弗细胞加1.0微克/毫升脂多糖会降低共培养肝细胞中的荧光强度。添加一氧化氮合成的选择性抑制剂NG-单甲基-L-精氨酸可显著抑制脂多糖激活的库普弗细胞诱导的肝细胞中罗丹明-123荧光的变化。脂多糖的门静脉输注也会降低灌注大鼠肝脏中罗丹明-123的荧光。中央周围区域的这种降低显著增强。持续输注脂多糖会引发NADH的自发荧光；这种反应在中央周围区域也会增强。我们用异硫氰酸荧光素标记的脂多糖表明肝小叶中注入的脂多糖的主要摄取部位在门静脉周围区域。我们的结果表明，线粒体能量代谢的抑制主要发生在远离脂多糖摄取部位的中央周围区域。持续给予NG-单甲基-L-精氨酸可显著减轻脂多糖诱导的罗丹明-123荧光降低和肝小叶NADH含量增加。（摘要截断于250字）
