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D J Tuma

Publications and source records attributed to D J Tuma.

At least 73 records · Page 4Linked to original sources
PubMed1991

Effects of alcohol on hepatic protein metabolism and trafficking.

Ethanol administration disorders protein trafficking in the liver. The protein secretory and plasma membrane assembly pathways have been shown to be impaired in the liver of ethanol-treated animals; however, traffic along the receptor-mediated endocytosis (RME) pathway appears to be especially susceptible to alterations by ethanol. Using asialoglycoproteins as model ligands for studying RME, we have identified at least three steps of this multi-step pathway that are affected by ethanol treatment. These altered steps are recycling of the receptor, internalization of the receptor-ligand complex and dissociation of the ligand from its receptor in endosomes. Ethanol-induced derangements of RME are more severe in the perivenule region, where alcoholic liver injury starts and predominates, than in the periportal region of the liver. Recent studies have shown that the endocytosis of other ligands, including epidermal growth factor and insulin, is also altered by ethanol treatment. Mechanisms which have been proposed to explain faulty RME include: acetaldehyde adducts to tubulin resulting in impaired microtubule function, improper acidification of endosomes and defective receptor clustering in coated pits. Since RME represents an important process by which levels of various hormones, growth factors and other ligands are regulated, and since RME may also be an integral process by which the biological effects of various ligands are elicited, changes in this important process could disrupt numerous metabolic and homeostatic events in the liver.

Alcoholism↗
PubMed1991

The chemistry of acetaldehyde-protein adducts.

Acetaldehyde reacts with a variety of proteins to form both stable and unstable adducts. Unstable adducts are readily reversible and are largely represented by Schiff bases. Stable adducts are essentially irreversible products that are characterized by their resistance to various treatments. The structures of stable adducts have not been established, but it appears that the epsilon-amino group of certain lysines and the amino group of N-terminal amino acids participate in aldehyde binding. Stable adducts can form in the absence of a reducing agent, such as sodium cyanoborohydride, and are distinctly different than the ethylated amino groups formed under reductive conditions. Upon prolonged incubation, stable binding of acetaldehyde results in the formation of fluorescent products, and Schiff bases serve as intermediates of these advanced stable products. It is likely that several acetaldehyde molecules and perhaps at least two amino groups participate in the formation of these stable fluorescent products. Such a reaction mechanism could account for both inter-and intra-molecular cross-linking of proteins. Furthermore, it is likely that stable adducts are represented by multiple products whose structures may vary, depending upon the particular target protein. A reaction mechanism involving an aldol condensation and generation of a crotonaldehyde Schiff base is proposed as an essential step in stable adduct formation.

Acetaldehyde↗
PubMed1991

Zonal differences in ethanol-induced impairments in receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes.

We have shown previously that ethanol-induced defects in receptor-mediated endocytosis of asialoorosomucoid occurred as early as 1 wk after ethanol feeding. This study was undertaken as an initial attempt to establish a possible role of defective receptor-mediated endocytosis in liver injury by investigating whether differences exist in the effects of ethanol on receptor-mediated endocytosis in hepatocytes isolated from different regions of the liver. Perivenule cells, present in the distal half of the liver, are thought to be more susceptible to ethanol-induced liver injury than are the periportal cells located in the proximal half of the liver acini. For these studies, we fed male Sprague-Dawley rats for 7 days with liquid diets containing either ethanol (36% of calories) or isocaloric carbohydrate. Perivenule and periportal hepatocytes were then isolated using a digitonin-collagenase perfusion method. In control animals, cells isolated from the perivenule region bound significantly more ligand than did cells from the periportal region. Amounts of ligand internalized and degraded were also greater in perivenule than in periportal cells in these animals. After ethanol feeding, cells isolated from both the perivenule and periportal regions bound significantly less ligand than their respective controls. This impairment in surface and total binding was more pronounced in perivenule than in periportal cells. Internalization and degradation of the ligand were also more adversely affected in the centrilobular region as shown by decreases of greater than 60% in perivenule cells and by only 20% to 30% in periportal cells of ethanol-fed animals compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Effect of chronic ethanol administration on the uptake and degradation of asialoglycoproteins by the perfused rat liver.

We have shown previously reduced binding, internalization, degradation and receptor-ligand dissociation during receptor-mediated endocytosis (RME) of 125I-asialoorosomucoid (ASOR) by hepatocytes isolated from rats fed ethanol for 4-6 weeks. In the present study, we investigated the effect of ethanol feeding on RME by using the intact perfused liver as a model. Male, Sprague-Dawley rats were fed a liquid diet containing either ethanol (36% of calories) or isocaloric carbohydrate. Receptor-mediated endocytosis of 125I-ASOR was then examined over a time course of perfusion. In all cases, clearance of the labeled glycoprotein was followed by a slower but steady appearance of acid-soluble products in the medium. Ethanol-fed animals had a significantly (P less than 0.01) slower rate of clearance of the labeled ligand from the circulating perfusate than did control animals. Impairment of ASOR surface binding and degradation in ethanol-fed animals was also demonstrated in this model. When we examined the subcellular distribution of labeled ligand after various times of perfusion, we found that in control livers, a shift of radiolabeled ligand from the subcellular fractions containing endosomes and plasma membranes to fractions containing lysosomes occurred, while significantly less ligand was shifted to the lysosomes of ethanol-treated rats. These results show that ethanol administration inhibits RME of ASOR in the isolated perfused liver model, thus confirming our earlier reported defects in isolated hepatocytes. In addition, transport of ligand along the intracellular RME pathway was also shown to be altered by ethanol treatment as indicated by the impaired movement of ASOR from the endosomal to the lysosomal compartment.

Administration, Oral↗
PubMed1990

Effect of chronic ethanol administration on total asialoglycoprotein receptor content and intracellular processing of asialoorosomucoid in isolated rat hepatocytes.

Chronic ethanol administration markedly impairs the process of receptor-mediated endocytosis (RME) of a representative asialoglycoprotein, asialoorosomucoid (ASOR), by the liver (Casey et al. (1987) J. Biol. Chem. 262, 2704-2710). Decreased surface binding was the major defect reported in our initial study, along with impaired internalization and degradation of 125I-ASOR in chronically-fed ethanol animals. In this study, we further characterized these impairments by examining the content of intracellular receptors and by investigating ligand processing directed by these intracellular receptors. Ethanol administration for 5-7 weeks decreased intracellular ASOR receptor content by 40%, a result which was confirmed by using both a ligand-binding assay and an antibody-binding assay. In addition to a decreased number of intracellular receptors, an impairment in intracellular processing of receptor-ligand complexes was identified. In ethanol-fed animals, dissociation of receptor-ligand complexes was decreased during steady-state conditions of endocytosis at 37 degrees C. Impaired receptor-ligand dissociation did not alter the fate of the ligand which was to undergo diacytosis (ligand recycling), but did appear to impair degradation of intracellular ligand. These results indicate that chronic ethanol administration decreases ligand binding due to a decreased number of receptors and impairs intracellular processing of ASOR in hepatocytes.

Alcoholism↗
PubMed1990

Chronic ethanol administration impairs receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes.

The effects of chronic ethanol administration on the receptor-mediated endocytosis of epidermal growth factor were studied in isolated rat hepatocytes. In initial experiments, it was demonstrated that significantly less ligand was bound by hepatocytes isolated from rats fed an ethanol liquid diet for 5 to 7 wk than by cells isolated from chow-fed or pair-fed controls. Reduced binding was shown to be primarily caused by a decreased number of surface receptors rather than by changes in receptor affinity. When hepatocytes were incubated at 37 degrees C in the presence of a large saturating concentration of epidermal growth factor (80 nmol/L), intracellular levels of the ligand were significantly lower in cells from the ethanol-fed animals. However, no effect on degradation of the ligand was observed under those conditions. A defect in the initial stages of receptor-ligand internalization was also indicated because less surface-bound ligand was internalized and subsequently degraded in cells from the ethanol-treated rats. When the endocytosis of a lower, more physiological concentration of the growth factor (0.5 nmol/L) was studied, both the uptake of ligand and its degradation were markedly impaired in hepatocytes from the ethanol-fed animals. These results indicate that chronic ethanol administration impairs the receptor-mediated endocytosis of epidermal growth factor by the liver. The major impairment appears to be a reduction of cell surface receptors; however, other steps of the endocytotic pathway also appear to be affected. These altered steps include defective receptor-ligand internalization and changes in intracellular processing of the ligand leading to decreased degradation.

Animals↗
PubMed1990

Microtubular response to colchicine in adult and fetal rat hepatocytes.

1. Colchicine and related anti-microtubular drugs impair plasma protein secretion from adult rat liver explants 2-3-fold more than from fetal tissue. 2. Indirect immunofluorescence microscopy of cultured adult and fetal hepatocytes demonstrated that hepatocytes of both ages contain large numbers of densely packed microtubules which are equally disassembled by 10 microM colchicine. 3. Colchicine (10 microM) reduced secretion of [14C]leucine-labelled proteins from cultured adult hepatocytes by about 50% but did not significantly impede fetal secretion. 4. These results confirmed that plasma protein secretion can proceed without an intact microtubular system in fetal hepatocytes.

Animals↗
PubMed1990

Methionine metabolism in the Buffalo rat following ethanol feeding.

Earlier studies have demonstrated that the Buffalo strain rat may have a defect in the process of methylneogenesis or lack the methyl-transfer enzyme system in the liver involved in methionine biosynthesis. The results of this study demonstrate that the enzyme systems responsible for methionine formation are present in the Buffalo rat and that these systems respond to ethanol feeding in a manner similar to that already shown in the Sprague-Dawley animal. These findings suggest that the previously described defect in methionine metabolism of the Buffalo rat may lie within the mechanism of methylneogenesis.

Animals↗
PubMed1990

Effects of ethanol on hepatic protein trafficking: impairment of receptor-mediated endocytosis.

Ethanol administration disorders protein trafficking in the liver. The protein secretory and plasma membrane assembly pathways have been shown to be impaired in the liver of ethanol-treated animals; however, traffic along the receptor-mediated endocytosis pathway appears to be especially susceptible to alterations by ethanol. Using asialoglycoproteins as model ligands for studying receptor-mediated endocytosis, we have identified at least three steps of this multi-step pathway that are affected by ethanol treatment. These altered steps are recycling of the receptor, internalization of the receptor-ligand complex and dissociation of the ligand from its receptor in endosomes. Ethanol-induced derangements of endocytosis are more severe in the perivenule region, where alcoholic liver injury starts and predominates, than in the periportal region of the liver. In addition, recent studies have shown that the endocytosis of other ligands, including epidermal growth factor and insulin, are also altered by ethanol treatment. Mechanisms which have been proposed to explain faulty endocytosis include: acetaldehyde adducts to tubulin resulting in impaired microtubule function, improper acidification of endosomes and defective receptor clustering in coated pits. Since receptor-mediated endocytosis by the liver represents an important process by which levels of various hormones, growth factors and other ligands are regulated, and since endocytosis may also be an integral process by which the biological effects of various ligands are elicited, changes in this important process could disrupt numerous metabolic and homeostatic events in the liver and total organism.

Alcohol Drinking↗
PubMed1990

Altered role of microtubules in asialoglycoprotein trafficking in developing liver.

Efficient receptor-mediated endocytosis of asialoglycoprotein by mature liver requires participation of microtubules that convey newly internalized ligand to lysosomes for degradation and receptor back to plasma membrane to continue endocytosis. To ascertain whether microtubular participation in asialoglycoprotein endocytosis is altered during development, we compared endocytosis of 125I-labeled asialoorosomucoid (ASOR) in neonatal rat hepatocytes to that in adult cells, with and without microtubular disruption by colchicine. Control experiments demonstrated that 125I-ASOR degradation in neonatal hepatocytes occurred at 70% of the adult rate during continuous endocytosis, although neonatal surface receptors were only approximately 40% as numerous. Colchicine disruption of microtubules reduced 125I-ASOR degradation and steady-state intracellular ASOR more in adults during continuous endocytosis. Degradation of 125I-ASOR prebound to surface receptors was equally impaired by colchicine in the two groups. Continuous ASOR endocytosis by colchicine-treated adult hepatocytes progressively depleted their surface receptors but minimally in neonates. Unlike colchicine, the protonophore monensin markedly impaired receptor recycling as well as postinternalization ligand trafficking in both neonates and adults. Thus these experiments demonstrate that asialoglycoprotein processing proceeds as efficiently in neonatal as in adult hepatocytes despite a reduced surface receptor population. Microtubules are required to maintain receptors on cell surface as well as for postinternalization trafficking in adult cells. During development, only the latter process substantially requires microtubules, indicating that microtubular participation in protein trafficking is selectively, not uniformly, diminished at this time in life.

Animals↗
PubMed1990

Role of microfilaments in asialoglycoprotein processing in adult and developing liver.

To assess the role of microfilaments in receptor-mediated endocytosis of asialoglycoproteins, hepatocytes isolated from adult and 6-day-old rats were treated with the antimicrofilamentous agent cytochalasin D and then incubated with 125I-asialoorosomucoid (ASOR). Cytochalasin D (50 microM) reduced degradation of continuously endocytosed ASOR (7.5 micrograms/ml) equally in adult and neonate to approximately 20% of control. Internalization of surface-bound ASOR suggested at least two discrete sites at which ligand translocation was inhibited by drug at both ages: 1) initial movement of receptor-ligand complex from cell surface to interior and 2) postinternalization ligand transit to lysosomes. Inhibition of plasma membrane translocation was confirmed by calculation of endocytotic rate constant (Ke) values, which were decreased to approximately 20-30% of control after cytochalasin D treatment. In contrast, the antimicrotubular drug colchicine did not reduce Ke values significantly nor did colchicine in combination with cytochalasin D impede lysosome-directed transport more than cytochalasin D alone. These results indicate that internalization of occupied asialoglycoprotein surface receptor is microfilament dependent irrespective of postnatal age and that subsequent participation of microfilaments in asialoglycoprotein trafficking is closely related to that of microtubules.

Actin Cytoskeleton↗
PubMed1989

Preferential covalent binding of acetaldehyde to the alpha-chain of purified rat liver tubulin.

Hepatic ethanol oxidation generates the reactive intermediate acetaldehyde, which binds to proteins. Previous work, using bovine brain tubulin as a model protein, has shown that acetaldehyde preferentially formed stable adducts on the alpha-chain of the heterodimeric molecule. This binding resulted in functional impairment of the tubulin/microtubule system as evidenced by a decreased ability of adducted tubulin to form microtubules. Since tubulin/microtubules are believed to be very important cytoskeletal components of the hepatocyte and results with brain tubulin were interesting, our goal was to extend these studies to liver tubulin. We purified tubulin from rat liver by a polymerization-based cycle method followed by phosphocellulose chromatography. We then characterized the covalent binding of [14C]acetaldehyde to liver tubulin. Naturally forming and cyanoborohydride-stimulated stable adducts formed linearly with liver tubulin in a manner almost identical to that with brain tubulin. We also found that the alpha-chain of the native heterodimeric liver tubulin molecule was the preferred site of adduct formation at low acetaldehyde to protein ratios. These results confirm and extend our previous findings with the brain tubulin model and further suggest that the alpha-chain of tubulin may be a preferential site for acetaldehyde-adduct formation during ethanol oxidation in the liver.

Acetaldehyde↗
PubMed1989

Increased covalent binding of acetaldehyde to calmodulin in the presence of calcium.

The regulatory protein, calmodulin, undergoes major conformational changes in response to changes in intracellular calcium concentration. Furthermore, calmodulin has been reported to have lysine residues which markedly increase their reactivity toward electrophilic substances in the calcium-loaded state. We found that calmodulin formed two to three times more stable adducts with acetaldehyde in the calcium-loaded state as compared to the calcium-free state. Competition-binding studies showed that calmodulin could preferentially compete with albumin for acetaldehyde in the presence, but not in the absence, of calcium. When calmodulin was in the calcium-loaded state, trifluoperazine, an inhibitor of calmodulin activity, significantly decreased the stable binding of acetaldehyde to the protein, whereas in the calcium-free state, minimal effects on binding were observed. Since calmodulin is involved in regulation of multiple important processes in the cell, it is possible that acetaldehyde-calmodulin adducts could contribute to liver injury by perturbation of calcium-dependent homeostatic mechanisms within the hepatocyte.

Acetaldehyde↗
PubMed1989

Covalent interactions of acetaldehyde with the actin/microfilament system.

The covalent binding of [14C]acetaldehyde to purified rabbit skeletal muscle actin was characterized. As we have found for other cytoskeletal proteins, actin formed stable covalent adducts under reductive and non-reductive conditions. Under non-reductive conditions, individual and competition binding studies versus albumin both showed that the G-form of actin is more reactive toward acetaldehyde than the F-form. When proteins were compared on an 'equi-lysine' basis under non-reducing conditions, G-actin was found to preferentially compete with albumin for binding to acetaldehyde. Time-course dialysis studies indicated that acetaldehyde-actin adducts become more stable with prolonged incubation at 37 degrees C. These data raise the possibility that actin could be a preferential target for adduct formation in cellular systems and will serve as the basis for ongoing studies aimed at defining the role of acetaldehyde-protein adducts in ethanol-induced cell injury.

Acetaldehyde↗
PubMed1989

Effect of chronic ethanol administration on protein catabolism in rat liver.

Hepatic protein catabolism was measured in rats which were pair-fed a liquid diet containing either ethanol or isocaloric maltose-dextrin (control diet). Within 12 days after initiation of pair feeding, the level of total hepatic protein in ethanol-fed rats was 26% higher than that in pair-fed control rats. During this time interval, the catabolic rates of both short-lived [3H]puromycin-labeled proteins and long-lived native [14C]bicarbonate-labeled proteins were measured in the two groups of animals. The degradation rate of short-lived [3H]puromycinyl proteins and peptides was the same in ethanol-fed and pair-fed control rats. However, the overall catabolic rate of long-lived proteins in rats fed the ethanol liquid diet for 2-10 days was 37-40% lower than that in pair-fed controls. This difference in protein turnover was not a general phenomenon, since the time-dependent decay of [14C]proteins in the hepatic microsome fraction of ethanol-fed rats was 33% slower than that in pair-fed controls, but the apparent rate of cytosolic protein catabolism was the same in both groups of animals. The differences in protein turnover did not reflect quantitative changes in lysosomal proteases since the activities of four hepatic lysosomal cathepsins were unaffected by alcohol administration. When rats were subjected to longer periods of pair feeding (16-25 days), the difference in overall hepatic protein catabolism between ethanol-fed rats and their pair-fed controls was considerably attenuated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Ethanol-induced impairments in receptor-mediated endocytosis of asialoorosomucoid in isolated rat hepatocytes: time course of impairments and recovery after ethanol withdrawal.

Chronic ethanol administration markedly impairs the process of receptor-mediated endocytosis (RME) of a representative asialoglycoprotein, asialoorosomucoid (ASOR), by the liver. In this study, we further characterized these impairments by identifying the time of onset for ethanol-induced changes in RME as well as establishing the time course for recovery to normal endocytotic values after ethanol withdrawal. Ethanol administration for 3 days did not alter any aspect of endocytosis examined in this study. After feeding ethanol to rats for 7 days, however, significant decreases in amounts of ligand bound, internalized, and degraded were apparent. These impairments persisted throughout the 5-week feeding study although the effects were somewhat attenuated with more prolonged ethanol feeding. In addition, an accumulation of intracellular receptors was observed in ethanol-fed animals relative to controls after 7 days of ethanol feeding. In all cases, recovery of endocytotic values to control levels was partially completed after 2 to 3 days of refeeding control diet and was fully completed after 7 days of refeeding. These results indicate that ethanol feeding for as little as 7 days profoundly impairs the process of RME by the liver. These impairments can be reversed after refeeding control diet for 7 days.

Animals↗