Influence of caveolin on constitutively activated recombinant eNOS: insights into eNOS dysfunction in BDL rat liver

Abstract

Diminished endothelial nitric oxide (NO) synthase (eNOS)-derived NO production from the hepatic vascular endothelium contributes to hepatic vasoconstriction in portal hypertension. The aim of this study was to examine the mechanism of this process by testing the influence of a constitutively active form of eNOS (S1179DeNOS) in both primary and propagated liver cells in vitro and in the sham and bile duct ligated (BDL) rat liver in vivo, using an adenoviral vector encoding green fluorescent protein (AdGFP) and S1179DeNOS (AdS1179DeNOS). AdS1179DeNOS transduction augmented basal and agonist-stimulated NO generation in nonparenchymal liver cells. Sham rats transduced in vivo with AdS1179DeNOS evidenced a decreased pressor response to incremental doses of the vasoconstrictor methoxamine compared with sham rats transduced with AdGFP. However, BDL rats transduced with AdS1179DeNOS did not display improved vasodilatory responses as evidenced by similar flow-dependent pressure increases to that observed in BDL rats transduced with AdGFP, despite similar levels of viral transgene expression. We next examined the influence of the eNOS inhibitory protein caveolin on S1179DeNOS dysfunction in cirrhotic liver. Immunogold electron microscopic analysis of caveolin in BDL liver demonstrated prominent expression not only in liver endothelial cells, but also in hepatic stellate cells. In vitro studies in the LX2 hepatic stellate cell line demonstrate that caveolin precipitates recombinant S1179DeNOS in LX2 cells, that recombinant S1179DeNOS coprecipitates caveolin, and that binding is enhanced in the presence of overexpression of caveolin. Furthermore, caveolin overexpression inhibits recombinant S1179DeNOS activity. These studies indicate that recombinant S1179DeNOS protein functions appropriately in normal liver cells and tissue but evidences dysfunction in the cirrhotic rat liver and that caveolin expression and inhibition in BDL nonparenchymal cells, including hepatic stellate cells, may account for this dysfunction.

A role for asymmetric dimethylarginine in the pathophysiology of portal hypertension in rats with biliary cirrhosis

“…Further support for this hypothesis is provided by the observation that eNOS gene transfer in rats with BDE-induced cirrhosis did not restore normal vasodilatory responsiveness, whereas it did in rats with CCl 4 -induced cirrhosis. 8,27 Because the level of eNOS enzyme was unaltered in our rats with biliary cirrhosis, the cause of the associated decreased NOS activity remains to be explained. Theoretically, eNOS malfunction might result from either a direct inhibition of eNOS or indirect through dysfunctional cofactors.…”

“…Theoretically, eNOS malfunction might result from either a direct inhibition of eNOS or indirect through dysfunctional cofactors. Shah and colleagues 19,27 provided support for the latter hypothesis by demonstrating that enhanced hepatic caveolin-1 protein levels in rats with BDE-induced cirrhosis may mediate reduced NOS activity, since NOS activity was partially restored when excess calmodulin, a protein that competitively binds eNOS, was added to the liver.…”

Inhibition of VEGF- and NO-dependent angiogenesis does not impair liver regeneration

“…NOS activity was measured as described previously (20). Briefly, detergent-soluble lysates were incubated for 20 min with a buffer containing 1 mM NADPH, 3 M tetrahydrobiopterin, 10 nM calmodulin, 0.25 mM CaCl 2, 10 M L-arginine, and 0.2 Ci L-[ 3 H]arginine at 37°C.…”

“…The reaction was terminated by the addition of 1 ml of cold stop buffer (20 mM HEPES, 2 mM EDTA, and 2 mM EGTA, pH 5.5), and the reaction mix was applied to a Dowex AG 50WX-8 resin column. Radiolabeled counts per minute of L-citrulline generation were measured and used to determine L-NAME-inhibited NOS activity (20).…”

Cirrhotic Ascites: Pathophysiological Changes and Clinical Implications

“…NO is thought to be the major vasodilator molecule in cirrhotic patients. The intrahepatic microcirculation is altered significantly in liver cirrhosis, secondary to both architectural and vasoactive humoral changes, resulting in an increase in vasoactive molecules associated with a decrease in intrahepatic NO production [38,39]. On the other hand, multiple studies have documented an elevated serum level of NO in the systemic and splanchnic circulation in both cirrhotic patients and in animal models [40][41][42][43].…”