Ethanol oxidation mediates impaired hepatic receptor-mediated enocytosis.
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Acetaldehyde↗
Biomedical subjects
D J Tuma
Publications and source records attributed to D J Tuma.
Effects of monensin on ethanol-induced alterations in function of hepatocellular asialoglycoprotein receptor subpopulations.
Chronic ethanol consumption is associated with multiple impairments in receptor-mediated endocytosis (RME) in hepatocytes. RME mediated by the asialoglycoprotein receptor seems to be especially impaired by ethanol. In the present study, we determined susceptibility of RME to alterations in ethanol-fed and pair-fed control animals by the addition of a carboxylic ionophore, monensin. Monensin inhibits acidification of prelysosomal vesicular compartments, which results in a decrease in the rate of receptor-ligand dissociation within the cell. Low levels (25 microM) of monensin have been shown to preferentially affect receptor-ligand dissociation of one subset (state 2) of asialoglycoprotein receptor, whereas dissociation in a second subset (state 1 receptors) seems to be relatively unaffected. We examined whether ethanol treatment preferentially affected one or another of these receptor subpopulations. Male Wistar rats were fed a standard ethanol-containing (36% of calories) or control diet for 10 to 14 weeks, and hepatocytes were prepared from the animals. Similar to previous results from our laboratory, surface and total ligand and antibody binding were decreased by 30 to 45% (p < 0.01) in ethanol animals, compared with controls. An ethanol-induced impairment of receptor-ligand dissociation was also apparent in these cells, as shown by increased ratios of bound-to-free ligand during continuous endocytosis. After monensin treatment, surface receptors on both ethanol and control cells showed a similar pattern of redistribution to the cell interior and intracellular inactivation. When kinetics of intracellular receptor-ligand dissociation were examined upon addition of monensin, the bound-to-free ligand ratios in both control and ethanol cells increased dramatically and to an equal extent. These results indicate that, in the ethanol animals, the pattern of sensitivity to monensin is unchanged relative to controls. Thus, the relative proportion of state 1 and state 2 receptor populations do not seem to be affected after long-term feeding, and ethanol may be a perturbant that affects both state 1 and state 2 receptor function.
Animals↗
Soluble proteins modified with acetaldehyde and malondialdehyde are immunogenic in the absence of adjuvant.
Recent studies have shown that the alcohol metabolites malondialdehyde and acetaldehyde can combine to form a stable adduct (MAA) on proteins. This adduct has been detected in the livers of rats chronically consuming ethanol, and serum antibodies to MAA have been observed at significantly higher concentrations in ethanol-fed when compared with pair-fed or chow-fed control rats. More recently, preliminary studies have strongly suggested that the MAA adduct is capable of stimulating antibody responses to soluble proteins in the absence of adjuvants. The antibodies produced recognize either the MAA epitope or the carrier protein itself. Therefore, it was the purpose of this study to examine the potential immunogenicity of MAA-modified exogenous proteins in the absence of adjuvants. Balb/c mice were immunized in the presence or absence of adjuvant with different concentrations of unmodified or MAA-modified proteins. The antibody response to both the MAA epitope and unmodified protein epitopes were determined by ELISA. In the absence of adjuvant, significant antibody responses were induced to both the MAA epitope and nonmodified protein epitopes. Smaller immunizing doses of MAA-protein conjugate favored the production of antibodies to nonmodified proteins, whereas larger doses induced a strong anti-MAA response. In studies to begin determining a mechanism for the specificity of the response in the absence of adjuvants, peritoneal macrophages were found to bind and degrade MAA-adducted proteins through the use of a scavenger receptor. This indicated that MAA-adducted proteins may be specifically taken up and epitopes presented to the humoral immune system in the absence of adjuvants. Importantly, these are the first data showing that an alcohol-related metabolite can induce an antibody response in the absence of adjuvant and suggesting a mechanism by which antibody to the MAA adduct or its carrier (exogenous or endogenous) proteins may be generated in vivo.
Acetaldehyde↗
Decreased binding and autophosphorylation of the epidermal growth factor receptor in ethanol-fed rats.
We have shown previously that binding and processing of epidermal growth factor are impaired in livers of ethanol-fed rats. In the current study, we examined these ethanol-induced alterations in greater detail by studying both high and low affinity epidermal growth factor binding as well as the ability of added ligand to stimulate receptor autophosphorylation. We also measured the binding of anti-receptor antibody to intact and permeabilized cells in order to determine more accurately the levels of receptor protein. Hepatocytes were isolated from ethanol-fed and pair-fed control rats. Ligand binding, antibody binding, and ligand-induced receptor autrophosphorylation were measured in the respective cell populations. In ethanol-fed animals, binding to both high and low affinity states of the hepatic epidermal growth factor receptor was decreased by 40-50% (P < 0.01). This ethanol-induced decrease in ligand binding was accompanied by a reduced ability of epidermal growth factor to stimulate receptor autophosphorylation (32% decrease, P < 0.01). In contrast, binding of anti-receptor antibody was not altered in ethanol-fed animals. In conclusion, chronic ethanol feeding decreased epidermal growth factor binding with a concomitant decrease in the ability of the receptor tyrosine kinase to phosphorylate tyrosine residues. These changes were not accompanied by an actual decrease in receptor protein content. These findings could be relevant to modified responses to this growth factor in the livers of chronic ethanol-fed animals.
Animals↗
Epitope characterization of malondialdehyde-acetaldehyde adducts using an enzyme-linked immunosorbent assay.
Malondialdehyde (MDA) and acetaldehyde react together with proteins in a synergistic manner and form hybrid protein adducts, designated as MAA adducts. In a previous study, a polyclonal antibody specific for MAA-protein adducts was used in an immunoassay to detect the presence of MAA adducts in livers of ethanol-fed rats. In the present study, the specific epitope recognized by the antibody was defined and the chemistry of MAA adduct formation was further characterized. When several synthetic analogs were tested for their ability to inhibit antibody binding in a competitive ELISA, the results indicated that the major determinant of antibody binding was a highly fluorescent cyclic adduct composed of two molecules of MDA and one of acetaldehyde. The structure of this adduct was shown to be a 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivative of an amino group of a protein. Examination of MAA adduct formation with a variety of proteins indicated that in addition to this specific fluorescent adduct, MAA adducts were also comprised of other nonfluorescent products. The amount of fluorescent epitopes present on a given protein was the major determinant of antibody binding as assessed in a competitive ELISA, although the efficiency of inhibition of antibody binding by these fluorescent epitopes on MAA-adducted proteins varied depending upon the particular protein. However, when these MAA-adducted proteins were hydrolyzed with Pronase, the concentration of these modified proteins necessary to achieve 50% inhibition of antibody binding in a competitive ELISA fell into a much narrower range of values, indicating that protein hydrolysis equalized the accessibility of the antibody to bind the epitope on these various derivatized proteins. In summary, a cyclic fluorescent adduct of defined structure has been identified as the epitope recognized by our MAA adduct antibody. In addition to this specific adduct, MAA adducts are also comprised of other nonfluorescent products.
Acetaldehyde↗
The effect of betaine in reversing alcoholic steatosis.
The feeding of ethanol to experimental animals results in fatty infiltration of the liver. Recent findings have shown that ethanol-induced steatosis is accompanied by a lowering in hepatic S-adenosylmethionine (SAM) levels. It is known that SAM provides substrates for reduced glutathione formation and offers the cell protection from toxic metabolic oxidants. A recent study in this laboratory demonstrated that dietary supplementation with betaine generated increased SAM in the liver and protected against ethanol-induced steatosis. The present study not only showed that betaine supplementation to rats protects the liver from alcoholic steatosis, but also demonstrated that once steatosis is established, treatment with betaine partially reversed the steatosis after cessation of ethanol feeding. Furthermore, this study indicated that betaine supplementation to the diet had the capacity to attenuate steatosis despite the continued feeding of ethanol.
Animals↗
Impairment of the asialoglycoprotein receptor by ethanol oxidation.
It is well established that ethanol exposure impairs the process of receptor-mediated endocytosis in hepatic cells, although the molecular mechanism(s) and the physiological consequence(s) of this impairment are unclear. Because addressing these mechanistic questions is difficult in vivo, we have developed a recombinant cell line of hepatic origin capable of metabolizing ethanol. In this study, we have used these recombinant cells, designated HAD cells, to investigate the ethanol-induced impairment to the receptor-mediated endocytosis of the hepatic asialoglycoprotein receptor. Comparing the binding of the ligand asialoorosomucoid in both the parental Hep G2 cells and the recombinant HAD cells, maintained in the presence and absence of ethanol, revealed decreased ligand binding in the HAD cells. This impairment was accentuated by prolonging the ethanol exposure, reaching approximately 40% in both surface and total receptor populations by 7 days. Addition of the alcohol dehydrogenase inhibitor pyrazole to the ethanol-containing medium abolished this impairment, indicating that the decreased binding was a result of the alcohol dehydrogenase-mediated oxidation of ethanol. Furthermore, using antibody specific to the asialoglycoprotein receptor, it was demonstrated that the ethanol-induced impairment in ligand binding was a consequence of decreased ligand binding and not a result of diminished receptor numbers. These results indicated that ethanol oxidation was required for the ethanol-induced impairment in ligand binding, and that the reduced ligand binding was a result of a decrease in the ability of the ligand to bind to the receptor.
Asialoglycoprotein Receptor↗
Decreased binding of asialoglycoproteins to hepatocytes from ethanol-fed rats. Consequence of both impaired synthesis and inactivation of the asialoglycoprotein receptor.
Chronic ethanol administration alters the process of receptor-mediated endocytosis in isolated rat hepatocytes. Using the asialoglycoprotein receptor (ASGP-R) as a model, we have previously shown decreased binding of asialoglycoproteins to this receptor after as early as 1 week of ethanol administration. In the present study, we further analyzed the mechanism(s) responsible for this impairment by determining the ligand and antibody binding characteristics of the ASGP-R in rats fed ethanol over a 5-week time course. The results presented here demonstrate that ethanol treatment for 4 days significantly impaired total ligand binding without affecting antibody binding. Ethanol administration for a longer period of 1-2 weeks resulted in intermediate impairments in both ligand and antibody binding. After 5 weeks of ethanol exposure, ligand and antibody binding were equally lowered. In contrast to total cellular receptor binding, surface binding of both ligand and antibody were decreased over the entire time course of ethanol administration. Our data indicate that the ASGP-R is initially inactivated during the time course of ethanol exposure and that a redistribution of surface receptors to intracellular compartments occurs. Northern blot analysis showed that there was a significant decrease in receptor mRNA content in the 5-week chronically fed animals but not in the animals fed for 1 week. In addition, after 5 weeks of ethanol feeding, biosynthetic labeling of the ASGP-R was decreased in the ethanol cells, indicating impaired synthesis of the ASGP-R. In summary, an early inactivation of the ASGP-R occurs during ethanol exposure followed by an actual decrease in protein and mRNA content for the receptor.
Animals↗
Acetaldehyde and malondialdehyde react together to generate distinct protein adducts in the liver during long-term ethanol administration.
Acetaldehyde and the lipid peroxidation-derived aldehyde malondialdehyde (MDA), are reactive compounds that are generated during ethanol metabolism in the liver, and both aldehydes have been shown to be capable of binding to proteins and forming stable adducts. Because similar concentrations of MDA and acetaldehyde can coexist in the liver during ethanol oxidation, protein adduct formation in the presence of both of these aldehydes was studied under both in vitro and in vivo conditions. When proteins were incubated in the presence of both MDA and acetaldehyde, MDA caused a marked and concentration-dependent increase in the stable binding of acetaldehyde to proteins. Maximum stimulation of binding occurred at approximately a fourfold molar excess of MDA relative to acetaldehyde when concentrations of 1.0 mmol/L and 0.1 mmol/L were tested. The formation of highly fluorescent product or products was associated with the MDA stimulation of acetaldehyde binding, indicating that new and distinct products were being generated. These hybrid adducts of MDA and acetaldehyde have been designated as MAA adducts. An affinity-purified polyclonal antibody was produced that specifically recognized MAA epitopes on proteins and did not cross-react with carrier proteins or proteins modified with either acetaldehyde or MDA alone. A quantitative competitive enzyme-linked immunosorbent assay (ELISA) was developed and detected the presence of MAA-modified proteins in liver cytosol from ethanol-fed rats but not in pair-fed controls. Quantification of the data from the competitive ELISA indicated the presence of approximately 75 pmoles protein-bound MAA per milligram liver cytosol proteins of the ethanol-fed animals. These results indicate that acetaldehyde and MDA can react together in a synergistic manner and generate hybrid adducts (MAA-adducts) and further suggest that MAA adducts may represent a major species of adducts formed in the liver during ethanol metabolism in vivo.
Acetaldehyde↗
Long-term ethanol administration alters the degradation of acetaldehyde adducts by liver endothelial cells.
Previous reports have shown that long-term ethanol administration alters receptor-mediated endocytosis (RME) of a variety of macromolecules by liver endothelial cells (LEC). Acetaldehyde is the major metabolic product of ethanol metabolism and has been shown to bind to proteins to form adducts. In this study, the level of protein modification by acetaldehyde necessary for the uptake and degradation of acetaldehyde-modified proteins by LEC was investigated. Bovine serum albumin (BSA) acetaldehyde adducts were prepared by incubation of albumin with acetaldehyde at 100 mmol/L for 1 hour at 37 degrees C, and 1 mmol/L or 0.2 mmol/L for 5 days at 37 degrees C. In situ liver perfusion in the presence of these adducts resulted in the degradation of 107 +/- 10.02 microg, 69.82 +/- 5 microg, and 2.5 +/- 0.42 microg of acetaldehyde-adducted albumin, respectively, during a 4-hour period. These values were decreased by 53%, 67%, and nearly 100%, respectively, in livers from ethanol-fed rats. Additionally, modification of protein with 1 mmol/L of acetaldehyde for different periods of time and/or pH altered the amount of 14C-acetaldehyde binding, but no significant changes in degradation were observed. Finally, an excess of formaldehyde-modified albumin totally inhibited the degradation of acetaldehyde adducts, suggesting that they use the same receptor. These data show that acetaldehyde-modified proteins may be taken up and degraded by the scavenger receptor on LEC. This uptake and degradation are dependent on the extent modification of the protein by acetaldehyde, and long-term ethanol consumption decreases the degradation of acetaldehyde-protein adducts.
Acetaldehyde↗
Effects of chronic ethanol administration on the endocytosis of cytokines by rat hepatocytes.
The effects of chronic ethanol administration on the endocytosis of three representative cytokines were investigated in isolated rat hepatocytes. When hepatocytes were isolated from rats that were fed an ethanol liquid diet for 12 to 13 weeks, these cells exhibited a decreased ability to internalize and degrade transforming growth factor-alpha, tumor necrosis factor-alpha and interleukin-6, compared with hepatocytes from the pair-fed controls. This impaired endocytosis of all three cytokines was accompanied by significant decreases in the amount of hepatocyte surface-bound cytokine. Changes in cytokine binding to surface receptors and reduced rates of receptor-cytokine complex internalization into the cells seem to be major contributors to defective endocytosis in hepatocytes from the ethanol-fed rats. Impaired hepatocyte endocytosis could lead to altered steady-state levels of cytokines in the liver and modified physiological responses to cytokines. These changes could affect homeostasis among the various cell types in the liver and could contribute to liver dysfunction and injury.
Animals↗
Zonal differences in ethanol-induced impairments in fluid-phase endocytosis in rat hepatocytes.
We have previously shown that ethanol administration impairs the processes of fluid-phase endocytosis (FPE) and receptor-mediated endocytosis (RME) in isolated rat hepatocytes after as early as 1 week of ethanol administration. The defects in RME were most prominent in the perivenule (PV) region of the liver lobule, the area wherein alcoholic liver disease has been shown to start and predominate. We undertook the present study to see if changes in FPE were likewise more apparent in the PV versus the periportal (PP) region of the liver. For these studies, we fed male Sprague-Dawley rats with an ethanol-supplemented liquid diet or an isocaloric control diet for 1 or 5 weeks. PV and PP hepatocytes were isolated using a digitonin-collagenase perfusion method. Internalization and efflux of the marker dye, Lucifer Yellow, was then examined in the cell populations. After as early as 1 week of feeding, cells from the PV region in ethanol-fed animals showed dramatic impairments in the net internalization of dye, compared with PV cells from the pair-fed controls, and these changes persisted throughout the 5-week feeding period. In contrast, internalization of Lucifer Yellow in cells from the PP region of the liver were not different between control and ethanol animals. Because net internalization represents the balance between uptake into the cells versus efflux from the cells, we examined these components individually. Early uptake of the dye into the cell was not altered by ethanol treatment. The decreased net internalization seemed to be caused by enhanced efflux of the dye, which was significantly increased in PV cells, compared with the same cell type in control animals. Cells from the PP region of the ethanol-fed animals did not exhibit altered efflux after either 1 or 5 weeks of feeding. These results indicate that ethanol-induced impairments in FPE are more dramatic in the PV region of the liver, and these impairments seem to result from an ethanol-induced enhancement of efflux.
Animals↗
Feeding patterns of rats chronically ingesting an ethanol-containing liquid diet.
We compared the feeding patterns of rats ingesting a 36% ethanol-containing liquid diet for 30 days with those of rats pair-fed an isocaloric liquid control diet or provided control diet or ground rat chow ad libitum. Ethanol-fed rats consumed fewer calories per day and gained less body weight than rats fed control diets ad libitum. Daily caloric intakes were approximately 50% lower during the first 10 days and 20% thereafter. Lower intakes in ethanol-fed rats occurred through a decrease in mean meal size rather than number of meals per day, although meals were more evenly distributed diurnally. Pair-fed rats ingested most of their food in one or two meals within a few hours of presentation. In a related experiment, a 4-hr duodenal infusion of ethanol at a rate comparable to that of ethanol ingestion resulted in plasma ethanol levels of 28 +/- 4 mM and suppressed 5-hr intake by approximately 40% by increasing the mean postmeal interval and satiety ratio. These results suggest that the suppressive effect of ethanol ingestion on food intake may be mediated in part by a post-gastric mechanism of ethanol action.
Alcoholism↗
Establishment of a recombinant hepatic cell line stably expressing alcohol dehydrogenase.
Hepatocytes cultured for extended periods of time lose the ability to express alcohol dehydrogenase and thus, the ability to efficiently oxidize ethanol. Therefore, it has been difficult to investigate the effects of chronic ethanol oxidation by hepatocytes in vitro. To circumvent this problem, we have inserted the coding region of an exogenous alcohol dehydrogenase gene into an hepatic cell line. Using the human hepatocellular carcinoma cell line, Hep G2, we have constructed an hepatic cell line that stably expresses alcohol dehydrogenase. These recombinant cells, termed HAD 73.1 cells, express approximately 40% of the alcohol dehydrogenase activity of freshly isolated rat hepatocytes. When the ethanol metabolizing ability of these cells was directly measured, the results indicated that not only were these cells able to metabolize ethanol at approximately 70% of the rate of freshly isolated rat hepatocytes but acetaldehyde concentrations of up to 50 microM were detected in the medium. Furthermore, the level of acetaldehyde produced during ethanol oxidation was augmented by cyanamide, an inhibitor of acetaldehyde oxidation, while the ability of these cells to metabolize ethanol was inhibited by pyrazole, an inhibitor of alcohol dehydrogenase. These results suggest that this in vitro system will be a valuable tool enabling detailed biochemical studies exploring the effects of chronic ethanol oxidation on the liver and the mechanisms of alcohol-induced hepatic cell injury.
Acetaldehyde↗
Effect of hyperosmolarity on both receptor-mediated and fluid-phase endocytosis in ethanol-fed animals.
We have shown previously that chronic ethanol administration impairs hepatic receptor-mediated endocytosis (RME) of asialoorosomucoid (ASOR), epidermal growth factor and insulin, whereas early uptake by fluid-phase endocytosis (FPE) of a fluorescent dye, Lucifer Yellow (LY), is not altered. Results of these studies suggested that ethanol-induced injury was primarily affecting endocytosis in coated pit areas of the plasma membrane while internalization in noncoated membrane areas was unaffected. In the present study, we investigated the effects of blocking clathrin-coated pit mediated endocytosis by hyperosmolarity on FPE of LY and on RME of ASOR. We also examined the effects of hyperosmolarity on the binding and internalization of insulin, a ligand endocytosed by both RME and FPE. Uptake of LY by noncoated regions of the membrane was not altered in control animals, whereas in hepatocytes from ethanol-fed animals uptake of LY was decreased by 35-40% in the presence of 0.12 M sucrose (P < 0.05). These hyperosmolar conditions almost completely inhibited (> 85%) the endocytosis of 125I-ASOR by RME in both ethanol and control cells. Results with insulin showed slight effects (20-30% impairment) on uptake of the ligand in the presence of sucrose. These results are consistent with previous reports that in normal cells the coated pit pathway is impaired by hyperosmolarity, whereas endocytosis in noncoated regions is unaltered. It appears, however, that both FPE and RME in hepatocytes from ethanol-fed animals are susceptible to perturbation by hyperosmolarity. These results indicate that the noncoated pit pathway may be sensitive to stressful conditions such as hyperosmolarity after ethanol treatment.
Animals↗
Specificity of N-ethyl lysine of a monoclonal antibody to acetaldehyde-modified proteins prepared under reducing conditions.
A monoclonal antibody has been developed that recognizes only protein-acetaldehyde (AA) adducts prepared under reducing conditions: 5 mM AA with 30 mM sodium cyanoborohydride overnight at 37 degrees. This monoclonal antibody is a mouse IgG2b that has been designated RT1.1. The primary adduct formed when proteins are exposed to acetaldehyde under reducing conditions is N-ethyl lysine (NEL). To examine the epitope specificity of RT1.1, inhibition ELISAs were developed using NEL and other possible inhibitors, such as arginine, ethylamine, lysine and proteins modified with AA under non-reducing conditions. RT1.1 (at half-maximum optical density, 50 ng/mL) was inhibited only by NEL and was independent of the carrier or the pH of the buffer used in the ELISA. Further evidence indicating that NEL is the epitope recognized by RT1.1 was obtained using mouse and human epidermal growth factor (EGF). Both proteins contain one alpha amino group but only the human-EGF contains lysine residues with epsilon amino groups. In experiments where these two proteins were modified with AA under reducing conditions, RT1.1 reacted only with human-EGF. These studies demonstrate that RT1.1 is specific for NEL that is formed by the ethylation of proteins with acetaldehyde under reducing conditions. Additionally, these studies demonstrate that the procedures and methods used herein may be useful for characterizing other antibodies prepared to AA-modified proteins under a variety of defined in vitro chemical conditions.
Acetaldehyde↗
Effect of chronic alcohol ingestion on hepatic folate distribution in the rat.
The mechanism by which ethanol impairs folate metabolism remains uncertain. In the present study, we used our new technique (affinity/HPLC) for folate analysis to study the effect of chronic alcohol ingestion on the content and distribution of folates in livers. Twelve male Sprague-Dawley rats (180 g) were divided into two groups, and fed for 4 weeks with Lieber-DeCarli semi-liquid isocaloric diets, with and without 5% ethanol. Livers were extracted in boiling, pH 9.3 borate buffers containing ascorbate/dithioerythritol. Folates in the supernatant fractions were purified by affinity chromatography and analyzed using ion pair high performance liquid chromatography. The data obtained showed that hepatic folate distribution in alcohol-treated rats differed from that of control animals in two ways. Livers from the ethanol-fed rats, when compared with those from control rats, exhibited increases in the percent concentrations of methylated tetrahydrofolates (21.46 +/- 2.21 vs 14.8 +/- 1.23), decreases in the percent concentrations of formylated tetrahydrofolates (25.62 +/- 4.02 vs 46.18 +/- 2.65) and higher concentrations of unsubstituted tetrahydrofolates (52.91 +/- 3.84 vs 38.88 +/- 2.50). In addition, alcohol ingestion was associated with longer glutamate chains of the folate molecules, characterized by lower relative concentrations of pentaglutamyl folates (29 vs 48%), and higher relative concentrations of hexa- and heptaglutamyl folates (55 vs 46% and 15 vs 6%) when compared with controls. The data are discussed in relation to the possibility that alcohol exerts its effect through: (1) inhibition of B12-dependent methyl transfer from methyltetrahydrofolate to homocysteine; (2) diversion of formylated tetrahydrofolates toward serine synthesis; and (3) interaction of acetaldehyde with tetrahydrofolates, thereby interfering with folate coenzyme metabolism.
5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
Long-term ethanol feeding selectively impairs the attachment of rat perivenous hepatocytes to extracellular matrix substrates.
BACKGROUND/AIMS: We have previously shown that long-term ethanol consumption by rats results in a profound decrease in hepatocyte attachment to various extracellular matrix substrates. The present study investigated whether differences in attachment exist between cells isolated from either the periportal or perivenous regions of the liver. METHODS: Rats received long-term ethanol, and hepatocytes were selectively isolated by the digitonin-collagenase perfusion method. The ability of periportal and perivenous cells isolated from ethanol-fed and pair-fed control rats to attach to plates coated with either laminin, fibronectin, or type I collagen was then assayed. RESULTS: With all substrates, the attachment of perivenous hepatocytes isolated from ethanol-fed animals was significantly impaired. Time-course studies showed that although the rate of attachment of perivenous cells from ethanol-fed animals was only slightly reduced, a dramatic decrease in absolute attachment was observed. Furthermore, the perivenous cells isolated from ethanol-fed animals detached more readily from the substrate-coated plates than the corresponding periportal cells or either periportal or perivenous cells from pair-fed controls. CONCLUSIONS: Long-term ethanol consumption impairs hepatocyte-extracellular matrix interactions more severely in the perivenous region of the liver. This finding could be relevant to the pathological changes observed in alcoholic liver injury.
Animals↗