Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, North Carolina
Submitted 1 July 2004 ; accepted in final form 20 April 2005
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ABSTRACT |
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endothelial nitric oxide synthase; alcoholic liver injury; protein interactions
In the liver, nitric oxide (NO) synthesized by the endothelial NO synthase (eNOS) isoform regulates hepatic blood flow and vascular resistance (30, 31). Numerous studies have shown that the production of NO is abnormal in the injured liver (18, 27, 32, 37). Shah et al. (32) have shown that, in response to incremental increases in blood flow, cirrhotic animals produced significantly less NO than control animals because of reduced eNOS activity. Similarly, NO release from sinusoidal endothelial cells is reduced in liver cirrhosis, owing to impaired function of endothelial cell NOS (31, 37). Furthermore, these data suggest that, at the cellular level, abnormal production of NO in injured liver contributes to elevated intrahepatic resistance and portal hypertension (27, 32, 37). However, whether reduced hepatic production of NO and NOS activity are associated with alcohol-induced liver injury remains unclear. There are, however, several indirect lines of evidence that support this possibility. Oshita et al. (25) showed that alcohol increases portal pressure, which was associated with hepatic hypoxia and necrosis. Furthermore, Kimura et al. (16) found that the production of NO in Kupffer cells was diminished by chronic alcohol administration. NO was expected to counteract the increase in hepatic vascular resistance elicited by ethanol, therefore reducing liver injury, which highlights a protective role for NO in alcohol-induced liver disease (25). In support of this notion, treatment with a NOS inhibitor [N-nitro-L-arginine methyl ester (L-NAME)] increased the severity of liver injury by alcohol, whereas L-arginine supplementation completely prevented liver injury by alcohol (22). Most, if not all, of the reported studies were undertaken in male rats, and none of the reported studies investigated the cellular mechanism implicated in alcohol-evoked reduction in hepatic NO production.
The main objective of the present study was to test the hypothesis that alcohol reduces NO production by inhibiting the expression and/or activity of hepatic eNOS in female rats. To test this hypothesis, we designed the present study to determine 1) whether chronic alcohol exposure reduces the hepatic NOS activity (NO production) in our model, and 2) if so, whether the reduced NOS activity is due to reduced expression of eNOS or due to abnormal association of eNOS with its regulatory proteins (caveolin-1 and calmodulin). We hypothesized that increased hepatic eNOS association with the inhibitory protein caveolin-1 or reduced association with the stimulatory protein calmodulin underlies ethanol-induced inhibition of eNOS catalytic activity. To this end, we performed a series of studies on the livers of rats that received liquid diet with or without alcohol [3% (vol/vol)] for 12 wk as an experimental model to test this hypothesis.
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MATERIALS AND METHODS |
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Blood ethanol concentration. Blood samples were obtained from conscious rats held in restrainers. A volume of 0.2 ml of blood was taken from a tail vein once per week. We measured the ethanol content of the samples using the enzymatic method described in a previous study from our group (6).
NOS activity assays. The NOS assay kit (Calbiochem, La Jolla, CA) was used to determine the NOS activity. Briefly, the liver tissue was homogenized by Polytron in a homogenization buffer (25 mM Tris·HCl, pH 7.4, 1 mM EDTA, and 1 mM EGTA). The tissue homogenate was centrifuged at 100,000 g for 60 min at 4°C. The pellet containing membrane-associated NOS was resuspended in homogenization buffer. The samples were incubated with a reaction solution (50 mM Tris·HCl, pH 7.4, 6 µM tetrahydrobiopterin, 2 µM flavine adenine dinucleotide, 2 µM flavin mononucleotide, 1.25 mM NADPH, and 0.75 µM CaCl2) and 1 µCi [3H]arginine at 37°C. To determine the NOS activity, duplicate samples were incubated for 30 min in the presence of 1 mM L-NAME or vehicle. The reaction was stopped by the addition of 400 µl of cold stop buffer (50 mM HEPES, pH 5.5 and 5 mM EDTA). Equilibrated resin (100 µl) was added into each reaction samples. After centrifugation, the radioactivity of elute was quantified. Radiolabeled counts per minute of L-citrulline generation were measured and used to determine L-NAME-inhibited NOS activity as in our previous study (35).
Immunoblotting. Liver tissue was homogenized in a homogenization buffer [50 mM Tris, pH 7.5, 0.1 mM EGTA, 0.1 mM EDTA, 2 µM leupeptin, 1 mM PMSF, 1% (vol/vol) Nonidet P-40, 0.1% SDS, and 0.1% deoxycholate]. After centrifugation (12,000 g for 5 min), protein was quantified in the supernatant by a modified Lowry assay procedure (the Bio-Rad protein assay system). For the Western blot analysis of eNOS, caveolin-1, calmodulin, and the protein (100, 40, and 40 µg, respectively) were loaded onto SDS-PAGE gels with appropriate concentrations (7.5, 12, and 12%, respectively) for the best separation. Proteins were transferred to nitrocellulose membranes after electrophoresis. After they were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS), the blots were incubated with the specified primary antibody [anti-eNOS dilution at 1:1,000 and anti-caveolin-1 dilution at 1:1,000 (Transduction Laboratories, San Diego, CA) and anti-calmodulin dilution at 1:1,000 (Santa Cruz Biotechnology, Santa Cruz, CA)] in TBS buffer containing 5% nonfat dry milk overnight at 4°C. After four washes, the blots were incubated with second antibody dilution in TBS containing 5% nonfat dry milk. After three additional washes in TBS with 0.1% (vol/vol) Tween 20, the blots were detected by enhanced chemiluminescence system and exposed to X-ray film as in our previous study (35).
Immunoprecipitation. Liver tissue was homogenized in a buffer containing 150 mM NaCl, 1% IGEPAL CA-630, 50 mM Tris, pH 8.0, 0.1 mM PMSF, and 1x protease inhibitor cocktail (Roche, Mannheim, Germany). After centrifugation (12,000 g for 5 min), protein was quantified by use of a modified Lowry assay procedure (the Bio-Rad protein assay system). The samples containing 5 mg of protein were precleared by incubation with protein A-Sepharose for 1 h at 4°C. Anti-eNOS polyclonal antibody (250 µg/ml) or rabbit IgG (3.5 µg, control) was added and mixed for 1 h at 4°C. A 50% protein A-Sepharose slurry was added to the mixture and incubated, with rotation, at 4°C overnight. The preparation of the immunoprecipitation was analyzed by Western blot analysis as described above.
Densitometric analysis. Densitometric analysis was performed on a video-based computerized system. Protein levels were quantified by measuring the optical density of the specific bands using the public domain NIH Image program version 1.60 (http://rsb.info.nih.gov/nih-image/). The NIH Image program has a built in "calibrate" command. This command allows us to transform pixel values captured directly by the camera, from a scale that is linear with respect to transmission, into an integrated optical density scale that correlates to concentration (33). Briefly, to calibrate the system, 1) the "Gel Plotting Macros" was chosen, 2) an appropriate coverage area was used to outline the first lane, 3) this same area was copied and pasted to outline each of the other lanes in succession, 4) the "Plot Lanes" command was used to generate the lane profile plots, and 5) the areas of the peaks were measured by clicking inside each one in succession with the wand tool. Finally, the measurements were recorded. Here, these measurements are presented as integrated optical density, which are related to the concentration in each measured lane. A stabilized fluorescent light box model B95 (Imaging Research, St. Catharines, Ontario, Canada) was used with a high-performance monochrome charge-coupled device camera model 495-2010 (COHU, San Diego, CA) to capture the image on the developed film. To achieve a reliable comparison between treatment and control results, all extracts from the treatment and control rats (n = 6 each) were assayed in the same Western blot. Furthermore, each extract was assayed at least two times. Therefore, the protein and immunoprecipitation data presented in the figures constitute the average (SE) of the individual values obtained from the alcohol-treated and control rats under the same experimental conditions.
Histological and immunohistochemical analyses. The liver tissue was obtained after the rat was euthanized by a fatal dose of pentobarbital sodium and after the liver was fixed in 4% PBS-buffered paraformaldehyde. The tissues were then transferred to 20% sucrose solution at 4°C until the tissues sank. The sucrose-infiltrated tissues were frozen and sectioned (10 µm) by microtome (HM 505E, Microm International). The hematoxylin and eosin staining for pathological examination was performed as reported (22). For immunohistochemical analysis, the sections (3 or 4 sections from each animal) were immunostained with anti-caveolin-1 polyclonal antibody (1:300, Transduction Laboratories) using a modification of the avidin-biotin-complex method following the manufacture's instruction (Vector Laboratories, Burlingame, CA). Sections from treatment and control groups (6 sections from each rat) were subjected to the same experimental conditions (incubation time, washings, and so forth). Control sections treated with the mouse IgG exhibited no positive staining (data not shown). Similarly, no positive staining was detected in sections treated with the primary or the secondary antibody alone (data not shown).
Immunofluorescence. We prepared the liver tissue cryostat sections (10 µm) using the method described above for histological analysis. Briefly, sections (6 from each rat) were blocked with normal goat serum, incubated with primary antibody overnight at 4°C, washed three times with PBS, incubated with the secondary antibody for 1 h at room temperature, and washed three times with PBS. The Golgi apparatus was identified by incubating the slices with the goat anti-ARAP1 antibody (Novus Biologicals, Littleton, CO) and 7-amino-4-methyl-coumarin-3-acetic acid (coumarin)-conjugated donkey anti-goat IgG (1:50 dilution; Jackson ImmunoResearch Laboratory, West Grove, PA). eNOS was detected with a polyclonal antibody (1:50; Transduction Laboratories) and a goat anti-rabbit-Texas red secondary antibody. For triple staining, caveolin-1 was identified with an anti-mouse monoclonal antibody (1:50; Transduction Laboratories) and a goat anti-mouse-FITC secondary antibody, whereas eNOS and the Golgi apparatus were detected as described above. The intracellular localization of these proteins was visualized on a Nikon E600 fluorescence microscope with appropriate filters and images captured with an Orca II charge-coupled device camera (Hamamatsu, Bridgewater, NJ) and analyzed with Metaview image analysis and acquisition software (Universal Imaging, Downingtown, PA). The color images presented are in pseudocolor. Control sections that were only incubated with the primary or secondary antibody showed no fluorescence (data not shown).
Statistical analysis. All data are presented as means ± SE. The two-tailed Student's t-test and ANOVA (time-course data) were used in the analysis of data with the level of significance set at P < 0.05.
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RESULTS |
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Effect of alcohol on expression of caveolin-1 and calmodulin in liver. As shown in the representative blot and the densitometry analysis (Fig. 3C), hepatic caveolin-1 expression was significantly increased in alcohol-treated compared with control rats. Comparable results were obtained from immunohistochemical study using the same antibody. The sections from control rats displayed weak granular positive staining (Fig. 2C). The sections from alcohol-treated rats showed stronger staining for hepatic caveolin-1 compared with that of control rats (Fig. 2D). In contrast to the increase in caveolin-1 expression (Fig. 3C), the calmodulin expression in the liver was not affected by chronic ethanol administration (Fig. 3D).
Colocalization of eNOS and caveolin-1 in hepatocytes. In this experiment, we investigated whether eNOS is trapped in the Golgi apparatus in hepatocytes and whether chronic ethanol administration influences 1) eNOS association with the Golgi membrane and 2) the colocalization of eNOS and caveolin-1 with the Golgi apparatus. Triple-label immunofluorescence analyses demonstrated the colocalization of eNOS and caveolin-1 with the Golgi apparatus in hepatocytes (Fig. 4). eNOS association with caveolin-1 was increased in hepatocytes of alcohol-treated compared with control rats (Fig. 4). Furthermore, as shown in Fig. 4, eNOS colocalization with the Golgi membrane was increased in hepatocytes of alcohol-treated compared with control rats.
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DISCUSSION |
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Whether the reduction in eNOS-derived NO contributes to, or is caused by, the liver injury in alcohol-treated rats cannot be ascertained from the present findings. It is likely, however, that the reduced eNOS-NO signaling in our model plays a contributory role in alcoholic liver injury for two reasons. First, liver injury observed in ethanol-fed rats was associated with significant reduction in eNOS catalytic activity. Second, reported evidence suggests that adaptive changes in NO synthesis in the liver confer a beneficial or protective role to counter the deleterious effects of ethanol on the liver. In support of this notion are the reported findings that treatment with a NOS inhibitor (L-NAME) increases the severity of liver injury, whereas treatment with the NO precursor, L-arginine, completely prevented the liver injury caused by alcohol (22). Furthermore, Kimura et al. (16) showed that the evoked production of NO by Kupffer cells is diminished by chronic alcohol administration. Together, these results suggested that NO is likely to be involved in alcohol-mediated modulation of the function of hepatic cells during alcohol intake. The mechanism by which hepatic NO confers protection against ethanol-induced liver injury is not fully understood. Suggested mechanisms include the following: 1) NO plays a significant role in stabilizing the hepatic microcirculation and protecting the liver from oxidative injury (22, 23); 2) a counterbalance exists in vivo between hepatic levels of NO and superoxide levels (2); and 3) NO counteracts TNF-dependent activation of caspase 3 (15), which plays a pivotal role in liver injury (5). Notably, TNF is known to be involved in alcoholic liver injury (5). Indeed, in alcohol-fed rats, the transition from steatosis to steatohepatitis is associated with an increase in the hepatic expression of TNF (4, 19). Thus the reduced catalytic activity of hepatic eNOS activity, which we observed in the present study, may contribute to the pathophysiology of alcohol-induced liver injury.
Another important finding from the present study is the differential effect of alcohol on the association of eNOS with its regulatory proteins, caveolin-1 and calmodulin. It is well established that the binding of eNOS with the ubiquitous calcium-regulatory protein calmodulin promotes NO production (28). By contrast, an association between caveolin-1, the structural protein of plasmalemmal caveolae, and eNOS leads to a reduction in the catalytic activity of the enzyme (8, 14). From these studies, a molecular paradigm has been proposed whereby NO production is regulated through a reciprocal and competitive interaction of calmodulin and caveolin with eNOS, with NOS activation enhanced by binding with calmodulin and inhibited by binding with caveolin-1 (14, 20). In this study, we show an enhanced and diminished binding of eNOS with caveolin-1 and calmodulin, respectively, along with the reduced NOS activity in the liver form alcohol-fed rats. A similar alteration in the interactions of eNOS with its regulatory proteins has been found in the other models of liver injury such as cirrhotic liver (32).
It was important to determine whether chronic alcohol feeding influences the hepatic expression of caveolin-1 or calmodulin protein to gain insight into the mechanism by which chronic ethanol feeding alters the association between hepatic eNOS and both proteins. We report, for the first time, an increase in the caveolin-1 protein level, but not calmodulin, in the livers of alcohol-fed rats. A prominent increase in hepatic caveolin-1 protein level has also been found in experimental models of liver disease, suggesting a pathophysiological role for caveolin-1 upregulation in the liver (10, 32). The mechanisms of ethanol-evoked enhancement of hepatic caveolin-1 expression remain unclear. It is possible that a link exists between increased cholesterol and subsequent increase in caveolin-1 elicited by ethanol in cultured cells (3, 12, 29), explaining, at least partly, the significant increase in hepatic caveolin-1 in our model. Interestingly, the hypolipidemic drug fenofibrate dose dependently reduced alcoholic fatty liver in rats fed ethanol in liquid diet (34); ethanol feeding paradigms used in the reported study (34) and the present study were very similar. Future studies are necessary to determine whether ethanol enhancement of caveolin-1 expression in liver involves lipid metabolism.
In endothelial cells, the majority of eNOS is found in the membrane fraction, predominantly trapped in the Golgi apparatus, with the rest in the plasma membrane (24). The organized spatial localization of eNOS on the caveolae in plasma membrane and Golgi complex in endothelial cells is considered an important regulator of its activity (9, 11, 21). We report a similar pattern in hepatocytes, where eNOS localization with caveolin-1 and the Golgi apparatus was evident in both alcohol-treated and control rats. However, eNOS localizations with caveolin-1 and with the Golgi membrane were increased in hepatocytes from alcohol-treated compared with control rats (Fig. 4). The mechanisms of ethanol-evoked enhancement of hepatic eNOS localization with caveolin-1 remain unclear. It is possible that the enhanced caveolin-1 expression in the liver of ethanol-fed rats contributed to the enhanced association between eNOS and caveolin-1 demonstrated by the immunoprecipitation and immunofluorescence/merging findings. Furthermore, reported studies suggest that morphological changes in endothelial cells may affect the interaction between eNOS and cell membrane, which may lead to the increased eNOS localization with caveolin-1 and ultimately to reduced enzyme inactivity (21). Finally, it is possible that impaired membrane function in hepatocytes of ethanol-treated rats (17) contributes to the altered spatial localization of eNOS in hepatocytes obtained from ethanol-fed rats in the present study.
To summarize, results of the present study demonstrate, for the first time, that the alcoholic fatty liver is associated with diminished hepatic NOS-derived NO in a model of ethanol-induced liver injury. The diminished catalytic activity of hepatic eNOS is a result of increased binding of hepatic eNOS with the inhibitory protein caveolin-1 and reduced binding with the facilitator protein calmodulin in alcohol-treated rats. Increased caveolin-1 production in the livers of alcohol-treated rats along with altered subcellular localization of eNOS may contribute to the enhanced binding between eNOS and caveolin-1, which ultimately leads to reduced eNOS activity. By contrast, calmodulin levels remained unaltered despite a significant reduction in its binding with eNOS in the livers of ethanol-fed rats. Our findings may help elucidate the molecular mechanism underlying the inhibitory effects of alcohol on hepatic eNOS activity, which seems to be implicated in alcoholic liver injury.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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