Decreased constitutive hepatic nitric oxide synthase expression in secondary biliary fibrosis and its changes after Roux-en-Y choledocho-jejunostomy in the rat

Background/Aims: Inducible nitric oxide synthase (iNOS) in the liver has been shown to increase after stimulation by different compounds. Its exact role in cirrhosis is unclear, but it is thought to be an important factor in the development of the hyperdynamic state. In contrast, little is known about the constitutive form of nitric oxide synthase (cNOS) in the liver, and in particular in liver disease. We therefore investigated immuno-histochemical expression of endothelial NOS (cNOS) and, in addition, of macrophage. NOS (iNOS), a constitute and inducible form of NOS, in secondary biliary fibrosis and after reversing these changes by biliodigestive anastomosis. Methods: Sprague-Dawley rats were allocated to one of seven groups: sham-operated controls (CTR), bile duct ligation for 3 weeks (BDL) and BDL followed by Roux-en-Y choledochojejunostomy (RY); the latter group was studied after 1, 2, 3, 7 and 45 days (RY1, RY2, RY3, RY7, and RY45). cNOS-positive hepatocytes were stained with a monoclonal antibody specifically recognizing this isoenzyme, and quantitated stereologically. Results: Virtually all hepatocytes were positive for cNOS in CTR (99.9±0.2%); this was markedly reduced in BDL (73.7±9.8%); p<0.05). After RY, cNOS positive hepatocytes increased to reach normal levels (99.7±0.5%) in RY45. In contrast to cNOS, there was no iNOS positive staining with a monoclonal macrophage NOS antibody for this isoenzyme, which was confirmed by determining total iNOS protein concentration by Western blot. Serum nitrite/nitrate concentrations mirrored these findings and decreased from 7.9±3.7 mol/l in CTR to 2.1±0.4 mol/l in BDL. After RY, nitrite/nitrate concentration increased to 22.7±30.1, 32.4±21.4 and 44.6±32.7 mol/l in RY1, RY2 and RY3, respectively; in RY45, serum nitrite/nitrate concentration was normal averaging 6.1±1.2 mol/l. Conclusions: The present investigation shows that, in secondary biliary fibrosis, endothelial nitric oxide synthase is decreased in hepatocytes, macrophage nitric oxide synthase is not increased, and that — in contrast to current thinking — nitrate/nitrate production is diminished.