Effect of chronic ethanol administration on hepatic eNOS activity and its association with caveolin-1 and calmodulin in female rats

Xu Wang and Abdel A. Abdel-Rahman

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


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Although chronic and excessive alcohol consumption is associated with liver disease, the mechanism of alcoholic liver injury is still not clear. Whether reduced hepatic production of nitric oxide, which is evident in models of liver injury, is associated with alcohol-induced liver injury has not been investigated. We measured nitric oxide synthase (NOS) activity in the liver of pair-fed rats receiving liquid diet with or without alcohol [3% (vol/vol)] for 12 wk. Compared with control rats, hepatic NOS activity was significantly reduced in alcohol-treated rats along with the evidence of liver injury. Interestingly, there was no difference in the hepatic expression of endothelial NOS (eNOS) between ethanol-fed and pair-fed rats. We then tested the hypothesis that an imbalance between the binding of eNOS with inhibitory and stimulatory proteins may underlie the reduced activity of eNOS because eNOS catalytic activity is regulated partly through dynamic interactions with the inhibitory protein caveolin-1 and the stimulatory protein calmodulin. We found that hepatic caveolin-1 was markedly increased in alcohol-treated rats compared with control rats, whereas calmodulin remained unaltered. The binding of caveolin-1 and calmodulin with eNOS was increased and decreased, respectively, in alcohol-treated rats. Our results suggest that chronic alcohol intake attenuates hepatic eNOS activity by increasing the expression of the inhibitory protein caveolin-1 and enhancing its binding with eNOS.

endothelial nitric oxide synthase; alcoholic liver injury; protein interactions


IT IS WELL ESTABLISHED THAT chronic and excessive alcohol intake is associated with liver disease (13, 36). Alcoholic liver disease is categorized mainly in three major pathological settings: alcoholic steatosis (alcoholic fatty liver), alcoholic hepatitis, and alcoholic fibrosis and cirrhosis (13, 36). The understanding of how alcohol damages the liver has expanded substantially over the past decade. Many factors have been implicated in alcohol-induced liver injury. However, the mechanisms underlying alcoholic liver disease are still unclear (1, 7, 26, 36).

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.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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Fourteen female Sprague-Dawley rats were used in the present study. The rats were obtained from Charles River (Raleigh, NC) at 11–12 wk of age (180–200 g). The rats were randomly separated into two groups. The first group was the ethanol treatment group: rats were fed the alcohol-containing liquid diet [3% (vol/vol) ethanol; Midwest Grain Products, Westoo, MO]. The second group was the control group: rats were pair-fed regular liquid diet. Diets were prepared fresh every other day and stored in the refrigerator until dispensed. Diet intake (ml/100 g) was measured every day, and the rats were weighed once per week. Rats were maintained on the alcohol or control diet for 12 wk. Experimental procedures were approved by the institutional animal care and use committee.

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.


    RESULTS
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Figure 1 shows the liquid diet daily intake, body weights, and blood ethanol concentration over the course of the experiment. The daily liquid diet intake was similar in the two groups of rats that subsequently received ethanol or was pair-fed and received isocaloric diet (week 0; Fig. 1). Compared with baseline data, the addition of alcohol [3% (vol/vol)] to the liquid diet caused a significant reduction in daily diet intake (week 1; Fig. 1). Alcohol-treated and control rats had similar baseline body weights, and there was a steady increase in body weights in both groups (Fig. 1). In the alcohol group, the average daily intake of alcohol was 6.1 ± 0.2 g/kg, and the blood alcohol concentration was relatively stable over the course of the study (Fig. 1). The average blood alcohol concentration was relatively low at 13.2 ± 1.6 mg/dl. Notably, blood sampling was performed early in the morning before rats were provided with the alcohol-containing liquid diet. This level of blood alcohol is comparable with the low levels during daytime in rats fed the same diet. Alcohol was not detectable in the blood of control group rats.



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Fig. 1. Liquid diet intake (A) and body weights (B) of the ethanol (EtOH)-fed and control rats (n = 6 each) over the 12-wk treatment period. C: weekly blood ethanol concentrations in the ethanol-fed rats.

 
Histological changes of the liver. To determine whether chronic alcohol intake caused liver injury, potential pathological changes in the liver were examined by hematoxylin and eosin staining. A comparison of liver sections obtained from rats fed alcohol or control diet revealed signs of fatty liver in the alcohol-fed rats, which demonstrated the ability of chronic (12 wk) alcohol feeding to produce pathological liver injury (Fig. 2, A and B).



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Fig. 2. A and B: representative sections from livers of a rat fed control diet (A) or a rat fed ethanol in liquid diet (B) for 3 mo. Fatty deposits (hematoxylin and eosin stain), consistent with liver injury, are evident in the liver sections obtained from the ethanol-fed rat. C and D: immunohistochemical detection of caveolin-1 in the liver of a control or alcohol-treated rat. Sections of liver tissue were obtained from alcohol-treated and control rats and fixed in 4% paraformaldehyde-PBS solution for immunohistochemical staining for caveolin-1. Sections from control rat livers (C) display diffused background with granular positive focal staining. Sections from alcohol-treated rat livers (D) show stronger staining for caveolin-1 compared with that of control rats. Solid lines in B and D represent 50 µm. Control sections treated with the mouse IgG or only primary or the secondary antibody exhibited no positive staining (data not shown).

 
Effect of alcohol on NOS activity and eNOS protein level in liver tissue. The membrane-associated NOS activity (conversion of radiolabeled L-arginine to L-citrulline) was significantly reduced in liver tissues of alcohol-treated rats compared with control rats (Fig. 3A). To investigate whether the decreased hepatic NOS activity in chronic alcohol-treated rats was the result of reduction of eNOS protein level, eNOS expression in liver tissue was examined by immunoblotting. As shown in the representative blot (Fig. 3B) and the densitometry analysis (n = 6 for each group; Fig. 3B), no significant difference in eNOS protein level was detected in alcohol-treated compared with control rats. Neither inducible NOS nor neuronal NOS protein was detected (Western blotting) in liver tissues of control or alcohol-fed rats (data not shown).



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Fig. 3. A: effect of chronic ethanol [3% (vol/vol)] intake for 3 mo on nitric oxide synthase (NOS) activity measured by L-[3H]citrulline generation in homogenized liver tissue. Membrane-associated NOS activity was significantly (P < 0.05) reduced in rats fed ethanol compared with rats fed control diet (n = 6 for each group). Hepatic endothelial NOS (eNOS; B), caveolin-1 (C), and calmodulin (D) protein expressions are also shown. Protein samples were prepared from liver tissues from rats fed ethanol or control diet. Densitometric data (n = 6 for each group) are presented as means ± SE. E: hepatic eNOS association with caveolin-1 or calmodulin in rats fed alcohol or control diet measured by immunoprecipitation (IP). Proteins were separated and analyzed by immunoblotting as detailed under MATERIALS AND METHODS. Association of eNOS with caveolin-1 and calmodulin was increased and decreased, respectively, in alcohol-treated compared with control rats. Results are means ± SE (n = 6 for each group). *P < 0.05 compared with the corresponding control value.

 
Effect of alcohol on the association of caveolin-1 and calmodulin with eNOS in liver. Whether the association of eNOS with caveolin-1 and calmodulin contributed to the reduction in hepatic NOS activity in the alcohol-treated rats was investigated by immunoprecipitation. Enhanced binding of caveolin-1 with eNOS occurred in liver tissues obtained from alcohol-treated compared with control rats (Fig. 3E). On the other hand, diminished calmodulin binding was evident in primary eNOS immunoprecipitates prepared from liver tissues of alcohol-treated compared with control rats (Fig. 3E).

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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Fig. 4. Colocalization of eNOS and caveolin-1 in Golgi apparatus in hepatocytes of alcohol-treated and control rats using triple staining and merging technique. Caveolin-1 was detected with an anti-mouse monoclonal antibody (1:50) and a goat anti-mouse-FITC secondary antibody (green). eNOS was detected with a polyclonal antibody (1:50 dilution) and a goat anti-rabbit-Texas red secondary antibody. The Golgi apparatus was detected with goat-polyclonal to ARAP1 and 7-amino-4-methyl-coumarin-3-acetic acid (coumarin)-conjugated donkey anti-goat IgG (1:50 dilution) (blue).

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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The cellular mechanisms implicated in NO production in injured liver constitute an important area of investigation because these processes may provide potential avenues for therapeutic intervention. The present study dealt with the chronic effects of alcohol on eNOS-NO signaling in the rat liver. The study was extended to investigate the effect of alcohol on two counterbalancing allosteric proteins, caveolin-1 and calmodulin, which modulate the activity of eNOS activity. We found that histological changes consistent with liver injury in alcohol-treated rats were associated with 1) a reduction in hepatic NOS activity (NOS-derived NO), 2) increased hepatic expression of caveolin-1, and 3) increased and decreased association of eNOS with the inhibitory protein caveolin-1 and the stimulatory protein calmodulin, respectively. The increased production of caveolin-1 and its enhanced association with hepatic eNOS may contribute to the reduction of hepatic NOS activity in alcohol-treated rats. These results provide the first analysis of molecular mechanisms underlying the alcohol-related regulation of eNOS activity in a rat model of alcoholic liver injury.

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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This study was supported by National Institute on Alcohol Abuse and Alcoholism Grants AA-07839 and AA-014441.


    ACKNOWLEDGMENTS
 
The technical assistance provided by Kui Sun and Dr. Wan-Xi Yang and Dr. Jian Zhang is appreciated.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. A. Abdel-Rahman, Dept. of Pharmacology and Toxicology, Brody School of Medicine, East Carolina Univ., Greenville, NC 27834 (e-mail: abdelrahmana{at}mail.ecu.edu)

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Arteel GE. Oxidants and antioxidants in alcohol-induced liver disease. Gastroenterology 124: 778–790, 2003.[CrossRef][ISI][Medline]
  2. Bautista AP and Spitzer JJ. Inhibition of nitric oxide formation in vivo enhances superoxide release by the perfused liver. Am J Physiol Gastrointest Liver Physiol 266: G783–G788, 1994.[Abstract/Free Full Text]
  3. Bist A, Fielding PE, and Fielding CJ. Two sterol regulatory element-like sequences mediate up-regulation of caveolin gene transcription in response to low density lipoprotein free cholesterol. Proc Natl Acad Sci USA 94: 10693–10698, 1997.[Abstract/Free Full Text]
  4. Bykov I, Jarvelainen H, and Lindros K. L-Carnitine alleviates alcohol-induced liver damage in rats: role of tumour necrosis factor-{alpha}. Alcohol Alcohol 38: 400–406, 2003.[Abstract/Free Full Text]
  5. Diehl AM. Nonalcoholic fatty liver disease: implications for alcoholic liver disease pathogenesis. Alcohol Clin Exp Res 25: 8S-14S, 2001.[CrossRef][ISI][Medline]
  6. El-Mas MM and Abdel-Rahman AA. Role of aortic baroreceptors in ethanol-induced impairment of baroreflex control of heart rate in conscious rats. J Pharmacol Exp Ther 262: 157–165, 1992.[Abstract]
  7. Fernandez Checa JC, Bellentani S, and Tiribelli C. Alcohol-induced liver disease: from molecular damage to treatment. Rev Med Chil 130: 681–690, 2002.[ISI][Medline]
  8. Garcia-Cardena G, Martasek P, Masters BS, Skidd PM, Couet J, Li S, Lisanti MP, and Sessa WC. Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the NOS caveolin binding domain in vivo. J Biol Chem 272: 25437–25440, 1997.[Abstract/Free Full Text]
  9. Garcia-Cardena G, Oh P, Liu J, Schnitzer JE, and Sessa WC. Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling. Proc Natl Acad Sci USA 93: 6448–6453, 1996.[Abstract/Free Full Text]
  10. Garver WS, Erickson RP, Wilson JM, Colton TL, Hossain GS, Kozloski MA, and Heidenreich RA. Altered expression of caveolin-1 and increased cholesterol in detergent insoluble membrane fractions from liver in mice with Niemann-Pick disease type C. Biochim Biophys Acta 1361: 272–280, 1997.[ISI][Medline]
  11. Govers R and Rabelink TJ. Cellular regulation of endothelial nitric oxide synthase. Am J Physiol Renal Physiol 280: F193–F206, 2001.[Abstract/Free Full Text]
  12. Hailstones D, Sleer LS, Parton RG, and Stanley KK. Regulation of caveolin and caveolae by cholesterol in MDCK cells. J Lipid Res 39: 369–379, 1998.[Abstract/Free Full Text]
  13. Ishak KG, Zimmerman HJ, and Ray MB. Alcoholic liver disease: pathologic, pathogenetic and clinical aspects. Alcohol Clin Exp Res 15: 45–66, 1991.[ISI][Medline]
  14. Ju H, Zou R, Venema VJ, and Venema RC. Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. J Biol Chem 272: 18522–18525, 1997.[Abstract/Free Full Text]
  15. Kim YM, Talanian RV, and Billiar TR. Nitric oxide inhibits apoptosis by preventing increases in caspase-3-like activity via two distinct mechanisms. J Biol Chem 272: 31138–31148, 1997.[Abstract/Free Full Text]
  16. Kimura H, Miura S, Higuchi H, Kurose I, Tsuzuki Y, Shigematsu T, Ebinuma H, Kato S, and Ishii H. Effect of chronic ethanol feeding on nitric oxide synthesis by rat Kupffer cells. Alcohol Clin Exp Res 20: 69A-72A, 1996.[Medline]
  17. Kurose I, Higuchi H, Miura S, Saito H, Watanabe N, Hokari R, Hirokawa M, Takaishi M, Zeki S, Nakamura T, Ebinuma H, Kato S, and Ishii H. Oxidative stress-mediated apoptosis of hepatocytes exposed to acute ethanol intoxication. Hepatology 25: 368–378, 1997.[ISI][Medline]
  18. Loureiro-Silva MR, Cadelina GW, and Groszmann RJ. Deficit in nitric oxide production in cirrhotic rat livers is located in the sinusoidal and postsinusoidal areas. Am J Physiol Gastrointest Liver Physiol 284: G567–G574, 2003.[Abstract/Free Full Text]
  19. Lu SC, Tsukamoto H, and Mato JM. Role of abnormal methionine metabolism in alcoholic liver injury. Alcohol 27: 155–162, 2002.[CrossRef][ISI][Medline]
  20. Michel JB, Feron O, Sase K, Prabhakar P, and Michel T. Caveolin versus calmodulin. Counterbalancing allosteric modulators of endothelial nitric oxide synthase. J Biol Chem 272: 25907–25912, 1997.[Abstract/Free Full Text]
  21. Murata T, Sato K, Hori M, Ozaki H, and Karaki H. Decreased endothelial nitric-oxide synthase (eNOS) activity resulting from abnormal interaction between eNOS and its regulatory proteins in hypoxia-induced pulmonary hypertension. J Biol Chem 277: 44085–44092, 2002.[Abstract/Free Full Text]
  22. Nanji AA, Greenberg SS, Tahan SR, Fogt F, Loscalzo J, Sadrzadeh SM, Xie J, and Stamler JS. Nitric oxide production in experimental alcoholic liver disease in the rat: role in protection from injury. Gastroenterology 109: 899–907, 1995.[CrossRef][ISI][Medline]
  23. Nishida J, McCuskey RS, McDonnell D, and Fox ES. Protective role of NO in hepatic microcirculatory dysfunction during endotoxemia. Am J Physiol Gastrointest Liver Physiol 267: G1135–G1141, 1994.[Abstract/Free Full Text]
  24. Ortiz PA and Garvin JL. Trafficking and activation of eNOS in epithelial cells. Acta Physiol Scand 179: 107–114, 2003.[CrossRef][ISI][Medline]
  25. Oshita M, Takei Y, Kawano S, Hijioka T, Masuda E, Nishimura Y, Fusamoto H, and Kamada T. Role of nitric oxide in ethanol-induced perturbation of hepatic microcirculation in rat liver. Alcohol Alcohol Suppl 2: 353–356, 1994.[Medline]
  26. Rambaldi A, Iaquinto G, and Gluud C. Anabolic-androgenic steroids for alcoholic liver disease. Cochrane Database Syst Rev 1: CD003045, 2003.[Medline]
  27. Rockey DC and Chung JJ. Reduced nitric oxide production by endothelial cells in cirrhotic rat liver: endothelial dysfunction in portal hypertension. Gastroenterology 114: 344–351, 1998.[ISI][Medline]
  28. Sessa WC. The nitric oxide synthase family of proteins. J Vasc Res 31: 131–143, 1994.[ISI][Medline]
  29. Shah V, Cao S, Hendrickson H, Yao J, and Katusic ZS. Regulation of hepatic eNOS by caveolin and calmodulin after bile duct ligation in rats. Am J Physiol Gastrointest Liver Physiol 280: G1209–G1216, 2001.[Abstract/Free Full Text]
  30. Shah V, Garcia-Cardena G, Sessa WC, and Groszmann RJ. The hepatic circulation in health and disease: report of a single-topic symposium. Hepatology 27: 279–288, 1998.[CrossRef][ISI][Medline]
  31. Shah V, Haddad FG, Garcia-Cardena G, Frangos JA, Mennone A, Groszmann RJ, and Sessa WC. Liver sinusoidal endothelial cells are responsible for nitric oxide modulation of resistance in the hepatic sinusoids. J Clin Invest 100: 2923–2930, 1997.[Abstract/Free Full Text]
  32. Shah V, Toruner M, Haddad F, Cadelina G, Papapetropoulos A, Choo K, Sessa WC, and Groszmann RJ. Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat. Gastroenterology 117: 1222–1228, 1999.[ISI][Medline]
  33. Shaw SL, Salmon ED, and Quatrano RS. Digital photography for the light microscope: results with a gated, video-rate CCD camera and NIH-image software. Biotechniques 19: 946–955, 1995.[ISI][Medline]
  34. Tsutsumi M and Takase S. Effect of fenofibrate on fatty liver in rats treated with alcohol. Alcohol Clin Exp Res 25: 75S–79S, 2001.[CrossRef][ISI][Medline]
  35. Wang X and Abdel-Rahman AA. Estrogen modulation of eNOS activity and its association with caveolin-3 and calmodulin in rat hearts. Am J Physiol Heart Circ Physiol 282: H2309–H2315, 2002.[Abstract/Free Full Text]
  36. Wang XD. Chronic alcohol intake interferes with retinoid metabolism and signaling. Nutr Rev 57: 51–59, 1999.[ISI][Medline]
  37. Yu Q, Shao R, Qian HS, George SE, and Rockey DC. Gene transfer of the neuronal NO synthase isoform to cirrhotic rat liver ameliorates portal hypertension. J Clin Invest 105: 741–748, 2000.[Abstract/Free Full Text]