Direct visualization of nitric oxide release by liver cells after the arrest of metastatic tumor cells in the hepatic microvasculature.
BACKGROUND
Our previous studies have shown that the injection of B16F1 melanoma cells into the mesenteric vein can induce the rapid local release of nitric oxide (NO) in the liver, causing apoptosis of the melanoma cells in the liver sinusoids and inhibiting the subsequent formation of hepatic metastases. In this study, we have investigated the distribution and cellular source of NO in this model.


METHODS
In situ liver perfusion was established in both wild-type (wt) and endothelial nitric oxide synthase knockout (eNOS KO) C57BL/6 mice. A specific fluorescent NO probe, 4,5-diaminofluorescein diacetate (DAF-2 DA) (5 micromol/L), was perfused into the portal venous system to label the liver tissue. Then, a MitoTracker Orange labeled B16F1 melanoma cell suspension (2 x 10(6) cells/ml) was injected through a portal vein catheter by a peristaltic pump. Images of the liver tissue were taken by confocal microscopy from a selected area to determine the cellular source of NO. For quantification, the fluorescence intensity of this area was measured over time by Fluoview software.


RESULTS
Diaminotriazolofluorescein (DAF-2T) fluorescence (indicating NO generation) was detected in hepatic parenchymal cells located in the periportal region in both wt C57BL/6 and eNOS KO C57BL/6 mice and was intensified by increased flow rate in the portal venous system. The B16F1 cells arrested in the periportal sinusoids, corresponding to zone 1 of the hepatic acinus. DAF-2T fluorescence was expressed by both sinusoidal lining cells and hepatocytes at the site of tumor cell arrest. The fluorescence intensity of these cells increased approximately 2-fold over a time of 500 s. In contrast, there was no increase in the fluorescence intensity of the sinusoidal lining cells and hepatocytes in mice perfused with buffer or in eNOS KO mice perfused with B16F1 cells.


CONCLUSIONS
This study demonstrates that NO is produced by hepatic parenchymal cells mainly located in the periportal zones and that the arrest of the B16F1 melanoma cells causes an eNOS-dependent local burst of NO by the sinusoidal lining cells and hepatocytes in the periportal areas.
Qi
Ke
K
Department of Pathology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.
Qiu
Hongming
H
Rutherford
John
J
Zhao
Yanshu
Y
Nance
Dwight M
DM
Orr
F William
FW
D000818:Animals; D045744:Cell Line, Tumor; D005260:Female; D005452:Fluoresceins / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D007091:Image Processing, Computer-Assisted; D008099:Liver / Q000378:metabolism* / Q000473:pathology; D008102:Liver Circulation; D008113:Liver Neoplasms / Q000473:pathology / Q000556:secondary*; D008545:Melanoma / Q000473:pathology / Q000556:secondary*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D009360:Neoplastic Cells, Circulating; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000172:deficiency; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D013314:Stress, Mechanical; D013997:Time Factors; D014018:Tissue Distribution
D005452:Fluoresceins; D005456:Fluorescent Dyes; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse