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Background & Aims: Arterial vasodilation plays a role in the pathogenesis of the complications of cirrhosis. This vasodilation is caused by the overproduction of arterial nitric oxide (NO). Bacterial translocation may be involved in NO synthase (NOS) up-regulation by activating both endothelial NOS (eNOS) and inducible NOS (iNOS). The prevention of intestinal gram-negative translocation by norfloxacin administration corrects systemic circulatory changes by decreasing NO production in cirrhosis. However, the signaling mechanisms for NO overproduction from bacterial translocation are unknown. In this study, we investigated the signal transduction pathway of bacterial translocation-induced aortic NOS up-regulation in cirrhotic rats., Methods: Proinflammatory cytokine levels, Akt and NOS activities, eNOS phosphorylation, and NOS expressions were assessed in aorta from norfloxacin-treated and untreated cirrhotic rats. Norfloxacin was administered to reduce intestinal bacterial translocation., Results: Aortic eNOS and iNOS protein expressions, Akt activity, and eNOS phosphorylation by Akt at serine 1177 were up-regulated in cirrhotic rats. Norfloxacin administration significantly decreased the incidence of gram-negative translocation and proinflammatory cytokine (tumor necrosis factor-alpha, interferon-gamma, and interleukin-6) levels; norfloxacin also decreased aortic Akt activity, eNOS phosphorylation, and NOS expressions and activities. The decrease in aortic Akt activity and NOS expressions also was obtained after colistin or anti-tumor necrosis factor-alpha antibody administration to cirrhotic rats., Conclusions: This study identifies a signaling pathway in which bacterial translocation induces aortic NOS up-regulation and thus NO overproduction in cirrhotic rats. These results strongly suggest that bacterial translocation and proinflammatory cytokines play a role in systemic NO overproduction in cirrhosis by the Akt pathway.

Background & Aims: In rats with portal vein stenosis, the initial cause of aortic nitric oxide (NO) overproduction might be overactivation of endothelial NO synthase (eNOS) related to increased shear stress. Cardiac output is higher in cirrhosis than in extrahepatic portal hypertension. The aims of this study were to evaluate the role of shear stress, vascular endothelial growth factor (VEGF), and cytokines in aortic eNOS up-regulation in rats with biliary cirrhosis and to compare these results with those in rats with portal vein stenosis., Methods: NOS activities, NOS protein, heat shock protein (Hsp) 90, and VEGF expressions were studied in rat aortas. Propranolol was administered to rats with cirrhosis to reduce cardiac output and thus shear stress., Results: In cirrhotic rats, the aortic eNOS protein was 3.0 and 1.7 times higher than in control and portal vein-stenosed rats, respectively. In cirrhotic rats, the Hsp90 content was 3.2 and 2.2 times higher than in control and portal vein-stenosed rats, respectively. Propranolol decreased NOS activity by 47% and eNOS and Hsp90 expression by 75% and 72%, respectively. Aortic VEGF expression was decreased in cirrhotic rats. VEGF-induced stimulation of NOS activity was greater in aortas from control rats than in aortas from portal vein-stenosed or cirrhotic rat aortas. eNOS expression was up-regulated after VEGF incubation. After lipopolysaccharide administration, eNOS expression did not change in portal vein-stenosed or cirrhotic rats., Conclusions: This study shows that in aortas from rats with biliary cirrhosis, systemic vasodilation depends mainly on eNOS up-regulation related to shear stress.

Background/aims: In vitro studies have shown that cirrhotic aortas are hyporeactive to the contractile effect of vasoconstrictors because upregulated endothelial nitric oxide-synthase (NOS) overproduces nitric oxide (NO). Although stimulation of endothelial small-conductance Ca2+-dependent K+ (SK(Ca)) channels may elicit vasorelaxation in normal arteries, the role of these channels in cirrhosis-induced hyporeactivity is unknown. Thus, the aim of the present study was to investigate the role of endothelial SK(Ca) channels in cirrhosis-induced, NO-mediated, in vitro aortic hyporeactivity to alpha1-adrenergic vasoconstrictors., Methods: Isolated thoracic aortas from cirrhotic and normal rats were used. The effects of apamin, a selective SK(Ca) channel blocker, were measured on the vascular reactivity to phenylephrine. In addition, SK(Ca) channel protein expression was studied. The effects of iberiotoxin and charybdotoxin, blockers of other K(Ca) channels, were also studied in cirrhotic aortas., Results: Apamin suppressed cirrhosis-induced aortic hyporeactivity to phenylephrine in an endothelium-dependent, NOS-inhibitor-sensitive manner. SK(Ca) channel protein was overexpressed in cirrhotic aortic walls. Iberiotoxin abolished cirrhosis-induced aortic hyporeactivity to phenylephrine in an endothelium-dependent but NOS-inhibitor-resistant manner. Charybdotoxin did not induce any significant increase in phenylephrine-elicited contraction., Conclusions: In cirrhotic aortas, the overexpression and overactivity of endothelial SK(Ca) channels contributes to in vitro NO-mediated hyporeactivity to the contractile action of alpha1-adrenergic agonists.

Background & Aims: In portal hypertension, the mechanisms responsible for nitric oxide (NO) overproduction and vasodilation have not yet been clearly identified. One hypothesis is that NO synthase (NOS) 3 is overactivated because of shear stress in endothelial cells caused by hyperkinetic circulation. The aim of this study was to evaluate aortic NOS3 after a reduction of blood flow by long-time beta-adrenoceptor antagonist administration., Methods: Propranolol or atenolol was administered by gavage in portal vein-stenosed and sham-operated rats. The vascular reactivity of thoracic aortic rings to phenylephrine, total aortic NOS activity, and aortic NOS3 messenger RNA and protein expressions were studied., Results: After propranolol or atenolol administration, the aortic hyporesponse returned to normal in portal vein-stenosed rats. Total aortic NOS activity was higher in portal vein-stenosed aortas and significantly decreased after beta-blocker administration. Aortic NOS3 expressions were more marked in portal vein-stenosed aortas than in controls, but NOS3 expressions were reduced after propranolol administration., Conclusions: In portal hypertension, aortic NOS3 activity and expressions are enhanced but return to normal after beta-blocker administration. These results suggest that in portal hypertension, increased shear stress, related to high blood flow, induces enhanced aortic NOS3.

Background/aims: Protein kinase C plays a role in the regulation of vascular cell contraction but its activity may be reduced by nitric oxide. In portal hypertension, the exact mechanism by which nitric oxide induces vascular hyporeactivity to vasoconstrictors is unclear. The aim of this study was to investigate the role of the interaction of nitric oxide and protein kinase C in the vascular reactivity in isolated aortae from portal vein-stenosed rats., Methods/results: The contractile response to phorbol 12,13-dibutyrate, a protein kinase C activator, was significantly reduced in portal vein-stenosed aortae compared to sham-operated aortae. Preincubation with N-nitro-L-arginine or endothelium removal enhanced the response to phorbol 12,13-dibutyrate. The hyporesponsiveness to phorbol 12,13-dibutyrate in portal vein-stenosed rat aortae was only corrected after endothelium removal. The time course of contractions induced by phorbol 12,13-dibutyrate showed that the contraction was maintained for 2 h in sham-operated aortae and decreased to baseline in portal vein-stenosed rat aortae. This decrease was inhibited by N-nitro-L-arginine preincubation or endothelium removal. Protein kinase C downregulation caused a more marked reduction of phenylephrine-induced contraction in portal vein-stenosed aortae than in sham-operated aortae. The time course of total nitric oxide synthase activity in the presence of phorbol 12,13-dibutyrate showed a decrease in nitric oxide synthase activity after 30 min in both groups. Nitric oxide synthase activity remained stable for 120 min in sham-operated aortae but returned to basal level in portal vein-stenosed aortae., Conclusions: Hyporeactivity to vasoconstrictors in portal vein-stenosed rat aortae may be due, in part, to a decrease in protein kinase C activation caused by nitric oxide overproduction.

Background & Aims: Although protein kinase C (PKC) alterations may play a role in the abnormal reactivity of cirrhotic rat aortas, its isoforms and cellular distribution are unknown. We therefore studied the protein expression and cellular distribution of PKC isoforms and their activation in cirrhotic rat aortas., Methods: Endothelium-denuded aortas from control and cirrhotic rats were examined. Immunoblots were performed with PKC isoform-specific antibodies. Aortic reactivity was determined for phorbol myristate acetate and phenylephrine after PKC down-regulation., Results: PKC-alpha expression was reduced in both the cytosolic and membrane fractions in cirrhotic aortas. Trace amounts of PKC-beta were detected in cirrhotic aortas. PKC-delta was detected in the cytosolic fraction of control and cirrhotic aortas. PKC-zeta was detected in the membrane fraction in control aortas and in the cytosolic fraction in cirrhotic aortas. Phorbol myristate acetate and phenylephrine triggered translocation of PKC-alpha and PKC-delta isoforms from the cytosol to the membrane in control aortas; in cirrhotic aortas, only PKC-alpha was translocated. Aortic reactivities were reduced after PKC down-regulation. PKC-alpha and -delta activities were reduced in cirrhotic aortas., Conclusions: These results suggest that a change in PKC isoforms may be responsible in part for the abnormal reactivity and intracellular transduction through the PKC pathway in cirrhotic rat aortas.

Hyporeactivity to vasoconstrictors in aortae from portal vein-stenosed rats is associated with an increased activity of endothelial NO synthase (NOS3). In contrast, during sepsis, which is common in cirrhosis, vascular hyporeactivity is associated with an induction of inducible NOS2. The aim of this study was to investigate the in vitro reactivity to phenylephrine and the regulation of NOS2 and NOS3 in aortae from portal vein-stenosed rats after lipopolysaccharide (LPS) administration. Aortic vascular reactivity for phenylephrine, aortic NOS activity, and NOS2 and NOS3 protein expression were determined 5 hours after intravenous LPS or saline administration. Moreover, aortic NOS activity was measured after 5-hour in vitro incubation in LPS. LPS induced a significantly smaller decrease in aortic tension in portal vein-stenosed than in sham-operated rats. Under baseline conditions, aortic NOS activity and NOS3 protein expression were higher in portal vein-stenosed than in sham-operated rats, and NOS2 protein expression was not detected in aortae from either group. After LPS administration, NOS activity and NOS2 protein expression increased significantly less in portal vein-stenosed than in sham-operated rat aortae. Similar results were obtained after in vitro incubation with LPS. Endothelium removal or NOS3 inhibition with the calmodulin inhibitor, W7, increased NOS activity in the aortae of portal vein-stenosed rats after LPS incubation. In conclusion, in aortae of portal vein-stenosed rats exposed to LPS, no further decrease in aortic reactivity to phenylephrine was observed, and the induction of NOS2 was down-regulated. Endothelium removal or calmodulin inhibition inhibits NOS3 overactivity and leads to normalized NOS2 activation after LPS in aortae from portal vein-stenosed rats.