Gregory Cadelina

Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat

BACKGROUND & AIMS A reduction in nitric oxide (NO) has been implicated as a cause of intrahepatic vasoconstriction in cirrhosis, but the regulatory mechanisms remain undefined. The aim of this study was to examine a contributory role for caveolin-1, a putative negative regulator of endothelial NO synthase, in mediating deficient intrahepatic NO production in the intact cirrhotic liver. METHODS Cirrhosis was induced by carbon tetrachloride inhalation. Flow regulation of NO production and perfusion pressure was examined in the perfused rat liver. Protein expression of endothelial NO synthase (eNOS), caveolin, and calmodulin was examined by Western blotting and immunohistochemistry. NOS activity and NO production were assessed by citrulline generation and chemiluminescence, respectively. Protein-protein interactions were examined using whole tissue protein immunoprecipitation. RESULTS In response to incremental increases in flow, cirrhotic animals produced significantly less NO(x) than control animals. NOS activity was significantly reduced in liver tissue from cirrhotic animals compared with control animals in the presence of similar eNOS protein levels. Deficient eNOS activity was associated with a severalfold increase in binding of eNOS with caveolin. Protein levels of caveolin-1 were markedly increased in the cirrhotic liver. CONCLUSIONS These studies provide evidence that enhanced expression and interaction of caveolin with eNOS contribute to impaired NO production, reduced NOS activity, and vasoconstriction in the intact cirrhotic liver.

Effect of Lactobacillus-Fermented Diets on Bacterial Translocation and Intestinal Flora in Experimental Prehepatic Portal Hypertension

Spontaneous bacterial infections in cirrhosis and portal hypertension have been attributed to trans- location of gut-derived bacteria, a process promoted by intestinal bacterial overgrowth and disruption of the gut mucosal barrier. Bacteriotherapy with Lactobacillus has been reported to correct bacterial overgrowth, stabilize mucosal barrier function, and decrease bacterial translocation in rat models of acute liver injury and failure. In this study we investigated the effect of Lactobacillus-supplemented diets on intestinal flora and on bacterial translocation rate in portal vein ligated rats. Lactobacillus-fermented milk (yogurt) containing at least 2 × 109 colony forming units/ml or placebo (water) was adminstrated by gavage twice daily (2 ml) for 9 days. Portal vein ligation was performed on day 7 of treatment. Bacterial translocation to mesenteric lymph nodes and quantification of intestinal flora was assessed by standard bacteriological cultures. Bacterial translocation was not significantly different between animals that received yogurt (82%) and those that received placebo (75%). Yogurt did not induce any significant changes in intestinal flora, whether it was produced with Lactobacillus acidophilus or Lactobacillus GG. In conclusion, in acute prehepatic portal hypertension, bacteriotherapy with Lactobacillus was unable to induce changes in bacterial translocation probably because it was unable to induce changes in bacterial flora.

Nitric oxide modulates both pre-sinusoidal and sinusoidal responses to phenylephrine in normal rat liver

Methods• Animals were studied 4 weeks after sham surgery or bile duct ligation (BDL). NOS activity was assessed by measuring L-NAME inhibited L-citrulline generation from liver lysates in the presence of a standard concentration ot calmodutin {0.1 /~M) or alternatively lO-fold excess in calmodulin (1.0 /~M). eNOS/iNOS protein levels were assessed by quantitative immunoprecipitation/Western blot analysis. Results, NOS activity was significantly reduced in liver lysates from BDL rat livers in the presence of 0.1 /~M calmodulin, the concentration routinely used in this assay [see Figure (white bars); *P<O.05; sham vs 6DL at 0.1 /~M calmodulin; n = 6]. Western blot analysis demonstrated no reduction in eNOS protein levels or iNOS protein expression in BDL liver lysates. Importantly, supplementation of lO-fold excess calmodulin (1.0/~M) did not affect NOS activity from sham animals, but significantly increased NOS catalytic activity in BDL lysates, thereby correcting deficient hepatic NOS activity (see Figure, **P<O.O5; 0.1 /LM VS. 1.0 ~M calmodulin in BDL; n=6). Conclusion. Provision of excess calmodulin corrects deficient NOS catalytic activity detected from BDL liver lysates in an experimental cell-free assay.