Temporal expression of hepatic inducible nitric oxide synthase in liver cirrhosis

AIM: Nitric oxide (NO) has been implicated in the pathogenesis of liver cirrhosis. We have found inducible nitric oxide synthase (iNOS) can be induced in hepatocytes of cirrhotic liver. This study further investigated the temporal expression and activity of hepatic iNOS in cirrhosis development. METHODS: Cirrhosis was induced in rats by chronic bile duet ligatjon (BDL). At different time points after the operation, samples were collected to examine NO concentration, liver function, and morphological changes. Hepatocytes were isolated for determination of iNOS mRNA, protein and enzymatic activity. RESULTS: Histological examination showed early cirrhosis 1-2 wk after BDL, with advanced cirrhosis at 3-4 wk. Bilirubin increased dramatically 3 d after BDL, but decreased by 47% on d 14. Three weeks after BDL, it elevated again. Systemic NO concentration did not increase significantly until 4 wk after BDL, when ascites developed. Hepatocyte iNOS mRNA expression was identified 3 d after BDL, and enhanced with time to 3 wk, but reduced thereafter. iNOS protein showed a similar pattern to mRNA expression. iNOS activity decreased from d 3 to d 7, but increased again thereafter till d 21. CONCLUSION: Hepatic iNOS can be induced in the early stage, which increases with time as cirrhosis develops. lts enzymatic activity is significantly correlated with protein expression and histological alterations of the liver, but not with systemic NO levels, nor with absolute values of liver function markers.     

Chronic administration of aminoguanidine reduces vascular nitric oxide production and attenuates liver damage in bile duct-ligated rats.

BACKGROUND: Nitric oxide (NO) has been implicated in the pathogenesis of liver cirrhosis. This study investigated the activity of nitric oxide synthase (NOS) in cirrhosis induced by bile duct-ligation (BDL) with NOS inhibitors. METHOD: Three days after operation, rats were randomized to receive aminoguanidine (AG, 25 mg/kg/day) or L-N(G)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg/day) for 21 days. RESULTS: Vascular NO production, which was increased in BDL cirrhotic rats, was reduced by 75% with AG but not L-NAME chronic administration. AG treatment attenuated liver damage, while L-NAME aggravated it. AG significantly suppressed inducible NOS (iNOS) expression in aorta of BDL rats at both mRNA and protein level, but much less efficient in reducing it in liver. In contrast, endothelial NOS (eNOS) expression was not markedly affected. Calcium-independent NOS activity, which was dramatically increased in aorta of BDL rats, was abolished by AG treatment. In liver, however, both calcium-dependent and -independent NOS activity were increased by AG treatment. CONCLUSION: Chronic administration of AG could reduce systemic NO levels as well as suppress iNOS expression and activity in aorta of BDL rats. It also improved liver function, possibly because of its ability to increase hepatic NOS activity, and to correct the systemic hemodynamic disorders by decreasing vascular NO production.     

Increased sensitivity to endotoxemia in the bile duct-ligated cirrhotic Rat.

Sepsis is a common complication of cirrhosis with a high mortality. In this study, we have investigated some of the pathways that may be involved in tissue injury and death. Bile duct-ligated (BDL) cirrhotic and control rats were challenged with lipopolysaccharide (LPS). Sensitivity to LPS was markedly enhanced in the BDL group, and was associated with increased liver injury and mortality. There was a 5-fold constitutive activation of nuclear factor kappa B (NFkappaB) in the liver of BDL rat controls (P <.001), and this was activated further, but to a similar extent, in the liver of both sham and BDL rats after injection of LPS. Plasma tumor necrosis factor alpha (TNF-alpha) increased more markedly in the BDL cirrhotic rats (2,463 +/- 697 pg/mL in BDL rats versus 401 +/- 160 pg/mL in the controls at 3 hours; P <.01). Plasma nitrite/nitrate concentrations were increased in the BDL controls at baseline, and increased further after LPS (P <.05), but did not differ from sham controls at 6 hours. Plasma F(2)-isoprostanes increased 6-fold in the cirrhotic rats and 2-fold in the controls (P <.01) indicative of lipid peroxidation. Esterified F(2)-isoprostanes in the liver increased 2- to 3-fold at 1 hour in control and BDL rats, but returned to baseline levels by 3 hours. Esterified F(2)-isoprostanes in the kidney increased by 2-fold in the BDL rats after LPS administration, but remained unchanged in sham controls. We conclude that there is a marked increase in sensitivity to LPS in BDL cirrhotic rats. This is associated with an enhanced TNF-alpha response and increased lipid peroxidation. These may be directly and causally related to mortality.     

Decreased renal expressions of heat shock protein-72 and -25 are associated with renal dysfunction in biliary cirrhotic rats.

During the course of liver cirrhosis, severe renal complications frequently occur. However, the pathogenesis of renal dysfunction in liver cirrhosis has not been completely understood. In this study, we investigated the association between renal function and expressions of renal heat shock proteins (HSPs) in biliary cirrhotic rats. Following bile duct ligation (BDL), renal function and expressions of HSPs were compared in control and BDL cirrhotic rats. Serum BUN and creatinine levels were significantly higher in cirrhotic rats compared with control rats at 4 weeks post-BDL operation. Renal expressions of HSP72 and HSP25 were decreased with progression of liver cirrhosis in BDL rats by Western blotting. Immunohistochemistry revealed that expression of renal HSP72 was suppressed in tubular epithelial cells, and expression of renal HSP25 was suppressed not only in tubular epithelial cells but also in blood vessels in rats with liver cirrhosis. Renal expressions of HSP90 and HSP60 did not differ between control and BDL rats. Renal function was impaired in biliary cirrhotic rats with decreased expressions of renal HSP72 and HSP25. These findings suggest that decreased expressions of renal HSP72 and HSP25 may be a part of the pathogenesis of renal dysfunction in liver cirrhosis.     

Endotoxemia produces coma and brain swelling in bile duct ligated rats

This study explores the hypothesis that the inflammatory response induced by administration of lipopolysaccharide (LPS) exacerbates brain edema in cirrhotic rats; and if so whether this is associated with altered brain metabolism of ammonia or anatomical disturbance of the blood-brain barrier. Adult Sprague-Dawley rats 4 weeks after bile duct ligation (BDL)/Sham-operation, or na?ve rats fed a hyperammonemic diet (HD), were injected with LPS (0.5 mg/kg, intraperitoneally) or saline, and killed 3 hours later. LPS administration increased brain water in HD, BDL, and sham-operated groups significantly (P < 0.05), but this was associated with progression to pre-coma stages only in BDL rats. LPS induced cytotoxic brain swelling and maintained anatomical integrity of the blood-brain barrier. Plasma/brain ammonia levels were higher in HD and BDL rats than in sham-operated controls and did not change with LPS administration. Brain glutamine/myoinositol ratio was increased in the HD group but reduced in the BDL animals. There was a background pro-inflammatory cytokine response in the brains of cirrhotic rats, and plasma/brain tumor necrosis factor alpha (TNF-alpha) and IL-6 significantly increased in LPS-treated animals. Plasma nitrite/nitrate levels increased significantly in LPS groups compared with non-LPS controls; however, frontal cortex nitrotyrosine levels only increased in the BDL + LPS rats (P < 0.005 versus BDL controls). CONCLUSION: Injection of LPS into cirrhotic rats induces pre-coma and exacerbates cytotoxic edema because of the synergistic effect of hyperammonemia and the induced inflammatory response. Although the exact mechanism of how hyperammonemia and LPS facilitate cytotoxic edema and pre-coma in cirrhosis is not clear, our data support an important role for the nitrosation of brain proteins.     

Ovariectomy Enhances Acetylcholinesterase Activity But Does Not Alter Ganglioside Content in Cerebral Cortex of Female Adult Rats

In the present work we investigated the effect of ovariectomy on acetylcholinesterase (AChE) activity and ganglioside content in cerebral cortex of female rats. We also studied the activity of butyrylcholinesterase (BuChE) in serum of these animals. Adult Wistar rats were divided into three groups: (1) naive females (control), (2) sham-operated females and (3) castrated females (ovariectomy). Thirty days after ovariectomy, rats were sacrificed by decapitation without anaesthesia. Blood was collected and the serum used for BuChE determination. Cerebral cortex was homogenized to determine AChE activity and extracted with chlorophorm:methanol for ganglioside evaluation. Results showed that rats subjected to ovariectomy presented a significant increase of AChE activity, but did not change the content and the profile of gangliosides in cerebral cortex when compared to sham or naive rats. BuChE activity was decreased in serum of rats ovariectomized. Our findings suggest that the alteration in the activity of brain AChE, as well as serum BuChE activity caused by ovariectomy may contribute to the impaired cognition and/or other neurological dysfunction found in post-menopausal women.     

Role of homocysteine and folic acid on the altered calcium homeostasis of platelets from rats with biliary cirrhosis

Previously, we have found that intracellular calcium homeostasis is altered in platelets from an experimental model of liver cirrhosis, the bile-duct ligated (BDL) rat; these alterations are compatible with the existence of a hypercoagulable state. Different studies indicate that cholestatic diseases are associated with hyperhomocysteinemia; thus, we hypothetized that it could contribute to those platelet alterations. In the present study, we have investigated the role of homocysteine (HCY) in platelet aggregation and calcium signaling in the BDL model. The effect of chronic folic acid treatment was also analyzed. Acute treatment with HCY increased the aggregation response to ADP and calcium responses to thrombin in platelets of control and BDL rats. Capacitative calcium entry was not altered by HCY. Chronic treatment with folic acid decreased platelet aggregation in control and BDL rats, but this decrease was greater in BDL rats. In folic acid-treated rats, thrombin-induced calcium entry and release were decreased in platelet of control rats but unaltered in BDL rats; however, capacitative calcium entry was decreased in platelets of control and BDL rats treated with folic acid. Reactive oxygen species were produced at higher levels by BDL platelets after stimulation with HCY or thrombin and folic acid normalized these responses. HCY plays a role in the enhanced platelet aggregation response of BDL rats, probably through an enhanced formation of ROS. Folic acid pretreatment normalizes many of the platelet alterations shown by BDL rats.     

Lack of detrimental or therapeutic effects of cyclooxygenase inhibition in bile duct-ligated rats with hepatic encephalopathy

BACKGROUND: The pathogenetic mechanisms of hepatic encephalopathy (HE) are not fully understood. Cerebral blood flow regulated by cyclooxygenase (COX) may be involved in the development of HE. There are no comprehensive data concerning the effects of COX inhibition on HE in chronic liver disease. METHODS: Male Sprague-Dawley rats weighing 240-270 g at the time of surgery were selected for experiments. Secondary biliary cirrhosis was induced by bile duct ligation (BDL). Those rats were then divided into two groups to receive i.p. injection of indomethacin (5 mg/kg per day) or distilled water for 7 days from day 36 to day 42 after BDL. The control group consisted of rats receiving a sham operation. Severity of encephalopathy was assessed by counts of motor activity. Plasma levels of tumor necrosis factor (TNF)-alpha and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), and liver biochemistry tests were determined after treatment. RESULTS: The motor activity in both groups of BDL rats were significantly lower than that of the control group (P < 0.001). As compared with the BDL rats treated with distilled water, BDL rats treated with indomethacin had significant lower levels of 6-keto-PGF(1alpha), but the motor activity, TNF-alpha levels and serum biochemistry tests were not significantly different between both BDL groups. CONCLUSIONS: Chronic indomethacin administration did not have significantly detrimental or therapeutic effects on the severity of encephalopathy in BDL rats.     

Ketorolac pharmacokinetics in experimental cirrhosis by bile duct ligation in the rat

The purpose of the present work was to study the pharmacokinetics of ketorolac, a poorly metabolized drug, in experimental cirrhosis. Cirrhosis was induced by bile duct ligation (BDL) for four weeks in male Wistar rats. Ketorolac was given intravenously (1 mg/kg ) or orally (3.2 mg/kg) to control (sham-operated) and BDL-rats. Determination of ketorolac in plasma was carried out by HPLC and estimation of pharmacokinetic parameters was performed by non-compartmental analysis. Indicators of liver damage and liver fibrosis were significantly increased (p < 0.05) in BDL compared to control rats. Experimental cirrhosis did not induce any significant alteration in intravenous ketorolac pharmacokinetics. Volume of distribution, clearance, AUC and t1/2 were similar in BDL and control animals. Notwithstanding, oral ketorolac bioavailability was significantly altered in BDL rats. AUC and Cmax were reduced, while tmax was prolonged, suggesting that both, the extent and the rate of ketorolac absorption were decreased. Results show that liver cirrhosis may result in significant pharmacokinetic alterations, even for poorly bio-transformed drugs, but that alterations may vary with the route of administration. In conclusion, uncritical generalizations on the effect of liver damage on drug kinetics should be avoided and systematic studies for every drug and every route of administration are thus recommended.     

Melatonin prevents increased asymmetric dimethylarginine in young rats with bile duct ligation

Abstract: Identifying and treating kidney injury in cirrhosis is important. Bile duct ligation (BDL) is a commonly used cholestatic liver disease model. We hypothesized that asymmetric dimethylarginine (ADMA) is involved in BDL‐induced oxidative stress and kidney injury, which can be prevented by melatonin. We also intended to elucidate whether increased ADMA is due to increased protein arginine methyltransferase‐1 (PRMT1, ADMA‐synthesizing enzyme) and/or decreased dimethylarginine dimethylaminohydrolase (DDAH, ADMA‐metabolizing enzyme). Three groups of young rats were studied, sham (N = 7), untreated BDL rats (N = 9), and melatonin‐treated BDL rats (N = 6, BDL + M). Melatonin‐treated BDL rats received daily melatonin 1 mg/kg/day via intraperitoneal injection. One‐third of the young BDL rats died compared with none in the BDL + M group. All surviving rats were killed 14 days after surgery. BDL rats had higher plasma aspartate aminotransferase, alanine aminotransferase, direct and total bilirubin, and ammonia levels than shams. They also had kidney injury characterized by increased tubulointerstitial injury scores and plasma creatinine and symmetric dimethylarginine levels, which melatonin prevented. Plasma ADMA levels were elevated in BDL rats, combined with increased hepatic PRMT1 and decreased renal DDAH activity. In addition, melatonin increased hepatic DDAH2 expression, increased DDAH activity and concomitantly decreased ADMA contents in both the liver and kidney. In conclusion, melatonin therapy decreased mortality and prevented kidney injury induced by BDL via reduction of ADMA (by increasing DDAH activity) and oxidative stress.     

Effect of hepatic iron concentration reduction on hepatic fibrosis and damage in rats with cholestatic liver disease

AIM: To assess the effect of iron reduction after phlebotomy in rats with "normal" hepatic iron concentration (HIC) on the progression of hepatic fibrosis, as a result of bile duct ligation (BDL). METHODS: Rats underwent phlebotomy before or after sham operation or BDL. Animals undergone only BDL or sham operation served as controls. Two weeks after surgery, indices of hepatic damage and fibrosis were evaluated. RESULTS: Phlebotomy lowered HIC. Phlebotomy after BDL was associated with body weight increase, lower hepatic weight, less portal hypertension, less periportal necrosis, less portal inflammation, lower hepatic activity index score and higher albumin levels. On the other hand, phlebotomy before BDL was associated with body weight decrease and hepatic activity index score increase. Phlebotomy after sham operation was not associated with any hepatic or systemic adverse effects. CONCLUSION: Reduction of HIC after induction of liver damage may have beneficial effects in BDL rats. However, iron deficiency could induce impairment of liver function and may make the liver more susceptible to insults like BDL.     

Increased vascular heme oxygenase-1 expression contributes to arterial vasodilation in experimental cirrhosis in rats

Vascular heme oxygenase (HO) regulates vascular tone in normal conditions and in some pathologic circumstances (e.g., sepsis). However, its possible role in the pathogenesis of arterial vasodilation in cirrhosis is unknown. To address this question, the expression and activity of HO in arterial vessels was studied in rats at 1, 2, and 4 weeks after bile duct ligation (BDL) or sham operation. A progressively increased expression of HO-1 was found in aorta and mesenteric arteries of BDL rats in a close chronologic relationship with the progression from acute cholestatic liver injury (1 week) to the fully developed cirrhosis with intense systemic arterial vasodilation (4 weeks). No changes were found in the expression of the constitutive isoform HO-2. HO-1 was mainly located in vascular smooth muscle cells of the arterial wall. Aortic HO activity increased in parallel with the expression of HO-1 (up to 600% in rats with cirrhosis compared with sham rats) and correlated with hemodynamic parameters. Increased expression of HO-1 and HO activity were also found in other organs, such as liver and spleen, though to a lesser extent compared with vascular tissue. The acute administration of an inhibitor of HO to cirrhotic rats, at a dose that normalized aortic HO activity, was associated with significantly greater effects on arterial pressure, total peripheral vascular resistance, and cardiac index, compared with effects in sham rats. In conclusion, these findings are consistent with a role for HO in the pathogenesis of arterial vasodilation in cirrhosis.     

Defective endothelial nitric oxide synthase signaling is mediated by rho-kinase activation in rats with secondary biliary cirrhosis

In liver cirrhosis, down-regulation of endothelial nitric oxide synthase (eNOS) has been implicated as a cause of increased intrahepatic resistance. We investigated whether Rho-kinase activation is one of the molecular mechanisms involved in defective eNOS signaling in secondary biliary cirrhosis. Liver cirrhosis was induced by bile duct ligation (BDL). We measured mean arterial pressure (MAP), portal venous pressure (PVP), and hepatic tissue blood flow (HTBF) during intravenous infusion of saline (control), 0.3, 1, or 2 mg/kg/hour fasudil for 60 minutes. In BDL rats, 1 and 2 mg/kg/hour fasudil significantly reduced PVP by 20% compared with controls but had no effect on HTBF. MAP was significantly reduced in response to 2 mg/kg/hour fasudil. In the livers of BDL rats, 1 and 2 mg/kg/hour fasudil significantly suppressed Rho-kinase activity and significantly increased eNOS phosphorylation, compared with controls. Fasudil significantly reduced the binding of serine/threonine Akt/PKB (Akt) to Rho-kinase and increased the binding of Akt to eNOS. These results show in secondary biliary cirrhosis that (1) Rho-kinase activation with resultant eNOS down-regulation is substantially involved in the pathogenesis of portal hypertension and (2) Rho-kinase might interact with Akt and subsequently inhibit the binding of Akt to eNOS.     

Endothelin-1 derived from spleen-activated Rho-kinase pathway in rats with secondary biliary cirrhosis

Aim: Splenectomy or partial splenic embolism has been reported to improve liver function in patients with hypersplenism and liver dysfunction. The aim of this study was to investigate the mechanism of improvement after splenectomy. Methods: Liver cirrhosis was induced by bile duct ligation (BDL). Rats underwent sham operation, splenectomy (Sp group), BDL, or BDL plus splenectomy (BDL + Sp group), and were subjected to experiments at 2 weeks after the operation. Portal venous pressure (PVP) and hepatic tissue blood flow (HTBF) were measured in each group. The plasma concentration of endothelin‐1 (ET‐1) and endothelial nitric oxide synthase (eNOS), RhoA and Rho‐kinase expressions were studied. Results: There were significant differences in PVP (17.9 ± 0.91 vs 23.3 ± 3.91 cmH₂O; P < 0.01) and HTBF (16.6 ± 1.72 vs 13.3 ± 1.82 mL/min; P < 0.01) between the BDL + Sp and BDL groups. In the liver of BDL rats, eNOS phosphorylation and NOx levels were decreased, accompanied by RhoA activation compared with the BDL + Sp group. Splenectomy decreased serum ET‐1 levels, RhoA activation and consequently increased eNOS phosphorylation. Conclusion: ET‐1 derived from the spleen might increase intrahepatic resistance by downregulating Rho signaling in liver cirrhosis. Splenectomy for splenomegaly in liver cirrhosis might partially improve liver function by enhancing intrahepatic microcirculation.     

Supplementation with vitamins E plus C or soy isoflavones in ovariectomized rats: effect on the activities of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase and cholinesterases

Since a previous study demonstrated that ovariectomized rats present an activation of Na⁺, K⁺-ATPase and acetylcholinesterase (AChE) activities, in the present study we investigated the influence of vitamins E plus C or soy isoflavones on the effects elicited by ovariectomy on the activities of these enzyme in hippocampus of ovariectomized rats. We also determined the effect of the same compounds on the reduction of serum butyrylcholinesterase (BuChE) activity caused by ovariectomy. Female adult Wistar rats were assigned to one of the following groups: sham (submitted to surgery without removal of the ovaries) and ovariectomized. Seven days after surgery, animals were treated for 30 days with a single daily intraperitoneous injection of vitamins E (40 mg/kg) plus C (100 mg/kg) or saline (control). In another set of experiments, the rats were fed for 30 days on a special diet with soy protein or a standard diet with casein (control). Rats were sacrificed after treatments and the hippocampus was dissected and serum was separated. Data demonstrate that vitamins E plus C reversed the activation of Na⁺, K⁺-ATPase and AChE in hippocampus of ovariectomized rats. Conversely, soy protein supplementation reversed the increase of AChE activity, but not of Na⁺, K⁺-ATPase activity, caused by ovariectomized group. Neither treatment was able to reverse the reduction of serum BuChE activity. Furthermore, treatments with vitamins E plus C or soy were unable to reverse the decrease in estradiol levels caused by ovariectomy. Our findings show that the treatment with vitamins E plus C significantly reversed the effect of ovariectomy on hippocampal Na⁺, K⁺-ATPase and AChE activities. However, a soy diet that was rich in isoflavones was able to reverse just the increase of AChE. Neither treatment altered the reduction in serum BuChE activity. Taken together, these vitamins and soy may have a protective role against the possible brain dysfunction observed in some menopause women. Vitamins E plus C and soy isoflavones may be a good alternative as a novel therapeutic strategy.     

Bone mineral metabolism and histomorphometry in rats with cholestatic liver disease

BACKGROUND: The etiology of osteopenia in cholestatic liver disease is uncertain. An animal model is needed in order to study the efficacy of therapeutic agents. AIMS: In order to characterise the bone disease in rats with cholestatic liver disease. METHODS: Four-month old male Sprague-Dawley bile duct-ligated (BDL) and sham-operated (SO) rats were studied. Twenty-eight days after surgery serum osteocalcin, a bone-formation marker, urinary deoxypyridinoline (DPD) cross-links, a resorption marker, and 25-hydroxyvitamin D3 were determined. Static and dynamic (tetracycline-based) histomorphometric analysis was performed on femurs and tibiae. RESULTS: All BDL rats developed biliary cirrhosis. Bile duct-ligated rats had lower bone mass, reflected in statistically significantly 13.5% lower femoral dry-weight, 16% lower femoral ash-weight, 42.7% lower tibial cancellous bone area and 19% lower trabecular thickness, compared with SO rats. Bile duct-ligated rats exhibited decreased bone formation manifested by statistically significantly 70% lower tetracycline double-labelling, 40% lower mineralising surface, 51% lower bone-formation rate and 47% lower osteocalcin compared with SO rats. Deoxypyridinoline levels were 20% lower in BDL rats. Bile duct-ligated rats had 52% lower serum 25-hydroxyvitamin D3 level, but no significant increase in cortical osteoid area. CONCLUSIONS: Bile duct-ligated rats develop osteopenia characterised by low bone-formation rate, and can be used for studying therapeutic agents for patients with cholestatic liver disease displaying similar bone changes.     

Vascular dysfunction in human and rat cirrhosis: role of receptor-desensitizing and calcium-sensitizing proteins

In cirrhosis, vascular hypocontractility leads to vasodilation and contributes to portal hypertension. Impaired activation of contractile pathways contributes to vascular hypocontractility. Angiotensin II type 1 receptors (AT1-Rs) are coupled to the contraction-mediating RhoA/Rho-kinase pathway and may be desensitized by phosphorylation through G-protein-coupled receptor kinases (GRKs) and binding of beta-arrestin-2. In the present study, we analyzed vascular hypocontractility to angiotensin II in cirrhosis. Human hepatic arteries were obtained during liver transplantation. In rats, cirrhosis was induced by bile duct ligation (BDL). Contractility of rat aortic rings was measured myographically. Protein expression and phosphorylation were analyzed by Western blot analysis. Immunoprecipitation was performed with protein A-coupled Sepharose beads. Myosin light chain (MLC) phosphatase activity was assessed as dephosphorylation of MLCs. Aortas from BDL rats were hyporeactive to angiotensin II and extracellular Ca2+. Expression of AT1-R and Galphaq/11,12,13 remained unchanged in hypocontractile rat and human vessels, whereas GRK-2 and beta-arrestin-2 were up-regulated. The binding of beta-arrestin-2 to the AT1-R was increased in hypocontractile rat and human vessels. Inhibition of angiotensin II-induced aortic contraction by the Rho-kinase inhibitor Y-27632 was pronounced in BDL rats. Basal phosphorylation of the ROK-2 substrate moesin was reduced in vessels from rats and patients with cirrhosis. Analysis of the expression and phosphorylation of Ca(2+)-sensitizing proteins (MYPT1 and CPI-17) in vessels from rats and patients with cirrhosis suggested decreased Ca2+ sensitivity. Angiotensin II-stimulated moesin phosphorylation was decreased in aortas from BDL rats. MLC phosphatase activity was elevated in aortas from BDL rats. Conclusion: Vascular hypocontractility to angiotensin II in cirrhosis does not result from changes in expression of AT1-Rs or G-proteins. Our data suggest that in cirrhosis-induced vasodilation, the AT1-R is desensitized by GRK-2 and beta-arrestin-2 and that changed patterns of phosphorylated Ca(2+) sensitizing proteins decrease Ca(2+) sensitivity.     

Morphological changes of rat astrocytes induced by liver damage but not by manganese chloride exposure

Liver cirrhosis is a common cause of death around the world. One of its more severe complications is hepatic encephalopathy. As a consequence of liver impairment, manganese (Mn) and other substances accumulate in the brain. Astrocytic morphological changes have been found in postmortem brains of cirrhotic patients. In this study we used a model of cirrhosis induced by bile duct ligation and Mn accumulation by exposing rats to MnCl₂ (1 mg Mn/ml) in their drinking water. Four experimental groups were used: Sham, Sham plus Mn treatment, BDL (bile duct ligated) and BDL plus Mn treatment. Brain Mn was measured by atomic absorption spectrophotometry in cortex, striatum and globus pallidus. Altered and normal astrocytes were counted in the same brain areas. Brain Mn was highest in rats of the BDLMn group. An increased number of altered astrocytes was found only in BDL groups, Mn did not modify this effect. No changes were found in the total number of astrocytes. According to our results, biliary obstruction induced an increase in the number of altered astrocytes since early stages of cirrhosis and Mn did not affect this effect.