(Received for publication, September 8, 1995; and in revised form, November 3, 1995 )
From the
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.
Receptor-mediated endocytosis (RME) ()is a process
common to many cell types including hepatocytes and has been shown to
be affected by ethanol
administration(1, 2, 3) . During RME,
molecules in the extracellular fluid bind to cell surface receptors and
are internalized as receptor-ligand complexes via a clathrin-coated
pit/coated vesicle pathway. Within this pathway, the vesicles lose the
clathrin coats, form endosomes, and are sorted in the acidic
compartment of uncoupling receptor and ligand. Once separated, ligands
and receptors can be routed to the lysosomes for degradation or they
can be recycled back to the cell surface(4) . The hepatic
asialoglycoprotein receptor (ASGP-R) is one of the well characterized
receptors that undergoes efficient recycling(5) . The ASGP-R
recognizes glycoproteins with terminal galactose or N-acetylgalactosamine residues and removes these potentially
harmful desialylated glycoproteins from the circulation(6) .
We have previously reported ethanol-induced impairments in the RME process of asialoorosomucoid (ASOR), a representative ligand for the hepatocyte-specific ASGP-R. Impairments in ligand binding, internalization, and degradation were demonstrated in hepatocytes isolated from animals fed ethanol for 5-7 weeks(1) . Decreased binding of ASOR by the hepatocytes of ethanol-treated rats appeared to be a major defect identified in the multi-step process of RME. The reduced binding was shown to be due primarily to a decrease in the number of functional cell surface receptors rather than to alterations of receptor affinity. Scatchard analysis of the binding data from animals fed for 5-7 weeks showed the presence of two classes of receptors, a high and a low affinity receptor. Ethanol administration decreased the number of both classes equally and did not alter the affinity of the receptor for ASOR in either class. In later studies we showed that decreased ligand binding occurred after as early as 1 week of ethanol administration(3) .
Surface and intracellular ASGP-R activity can be modulated by a variety of agents(7, 8) , and the receptors have been shown to undergo an intracellular inactivation-reactivation cycle that is necessary for proper receptor recycling and receptor-ligand dissociation(9) . The purpose of the present study was to clarify whether the decreased ligand binding we reported after ethanol administration was attributed to an actual reduction in the number of receptors (decreased receptor content) or to an inability of the receptors to recognize ligand due to receptor inactivation. For these experiments, ligand (ASOR) and anti-rat ASGP-R antibody binding were performed using both intact and permeabilized hepatocytes isolated from animals fed alcohol over a time course. Ligand binding provides a measure of receptor ``activity'' while antibody binding is more reflective of receptor ``content,'' since the antibody recognizes more than the ligand binding domain. We chose to examine feeding periods of a short duration of 4 days, an intermediate period of 1-2 weeks, and a longer feeding time of 5-7 weeks. In addition, analysis of messenger RNA levels after 1 and 5 weeks of feeding and in vitro biosynthesis of the ASGP-R was determined in these cells.
Hepatocytes were obtained from the livers of the treated animals by a modified collagenase perfusion technique as described previously by our laboratory(1) . The isolated hepatocytes were equilibrated in Krebs-Ringer buffer for 30 min at 37 °C and then incubated at 4 °C for the subsequent binding experiments and 37 °C for metabolic labeling studies. Additional cell aliquots were washed in cold phosphate-buffered saline, and the pellets were stored at -70 °C for future Western blot analysis and mRNA isolation.
Polyclonal anti-ASGP-R
antibody binding to isolated hepatocytes was likewise performed with
the intact and permeabilized cells. For these assays, rabbit polyclonal
serum to the ASGP-R at a dilution of 1:200 was added to the cells and
incubated at 4 °C for 60 min. After the binding period, the cells
were washed twice with Krebs-Ringer buffer, 1.5% BSA to remove unbound
antibody, after which fresh Krebs-Ringer buffer media containing I-protein A (6 µg/ml) was added; the incubation then
continued for another 60 min. Nonspecific surface and total binding
were determined by the amount of radioactivity measured in the presence
of nonimmune serum in the incubation media. Nonspecific binding was
routinely <10% of specific binding for both ligand and antibody
binding assays.
The incorporation of [S]methionine into newly
synthesized ASGP-R was performed as described by Schwartz and
Rup(21) . Briefly, hepatocytes isolated from control and
ethanol-fed animals were incubated with 200 µCi/ml of
[
S]methionine for periods of 0-90 min, and
1.0-ml aliquots were removed at each time point. The washed cell
pellets were initially solubilized and placed over a lactose column,
and the eluted receptor fraction was then subjected to
immunoprecipitation as described below.
In the first series of experiments, we determined the ability
of I-ASOR and a rabbit antibody generated against the rat
ASGP-R to bind to total cellular receptors (comprised of surface plus
intracellular receptors). By using both of these methods, we could
determine ethanol-induced alterations in receptor activity (reflected
by changes in ligand binding) versus alterations in receptor
content (reflected by alterations in antibody binding). Hepatocytes
were treated with low levels of digitonin (0.055% final concentration)
to permeabilize cells and make intracellular receptors available for
ligand and antibody binding. Use of this procedure to quantitate
cellular binding of
I-ASOR has been described previously
by our laboratory(2) . Our results show that after 4 days of
feeding, binding of ASOR to permeabilized cells was decreased
significantly (22%) in ethanol-fed animals when compared to controls (Fig. 1A). In contrast, no difference in binding of
anti-ASGP-R antibody between cells from control and ethanol-fed animals
was observed (Fig. 1B). These results indicated an
initial inactivation of the ASGP-R during the early 4-day feeding
period. After the chronic 5-7-week feeding period, both ligand
and antibody binding were equally decreased by an average of 50%,
reflecting a decrease in receptor content after long-term ethanol
feeding. The intermediate period of 1-2 weeks of feeding showed
that total ligand binding was decreased by 40%, but antibody binding
was reduced by only 25% in ethanol animals when compared to controls (Fig. 1, A and B). Thus, on the basis of the
ligand and antibody binding assays, it appears that during the early
stages of chronic ethanol consumption the ASGP-R is inactivated, and
after long-term ethanol feeding the actual content of the receptor has
been lowered.
Figure 1:
Determination of total cellular ASGP-R
activity and content by ligand and antibody binding after a time course
of ethanol administration. Isolated hepatocytes were obtained by a
collagenase perfusion technique from animals pair-fed for 4 days,
1-2 weeks, and 5 weeks. The binding of I-ASOR (A) and anti-ASGP-R antibody followed by
I-protein A (B) to total cellular ASGP-R was
performed on the permeabilized cells as described under
``Materials and Methods.'' Results are expressed as
femtomoles bound per million cells for control (
) and ethanol
(
) cells and are means ± S.E. for six determinations.
Values that are significantly different from control are indicated (*, p < 0.05).
In agreement with the data for antibody binding,
Western blot analysis confirmed these changes in receptor protein
content. For these experiments hepatocytes were lysed, subjected to
SDS-PAGE, and probed with anti-rat ASGP-R antibody and I-protein A. After 4 days of ethanol feeding, no change
in the amount of major 42-kDa subunit of the ASGP-R was observed, while
after 5 weeks of feeding, levels of the receptor were significantly
lowered (Fig. 2, A and C). Intermediate
decreases in protein content for the receptor were seen after the
1-2-week feeding period (Fig. 2B), similar to
data obtained with permeabilized cells. These data confirm that
receptor content is decreased after the later period of ethanol
administration.
Figure 2:
Immunochemical detection of the total
ASGP-R content. Hepatocytes were obtained from ethanol and control
pair-fed rats after 4 days (A), 1-2 weeks (B),
and 5 weeks (C) of ethanol administration. A 30-µl aliquot
of a 1.0 10
cell/ml suspension of control (C) and ethanol (E) cells was loaded into the gel
well, and the proteins were resolved by SDS-PAGE followed by transblot
analysis as described under ``Materials and Methods.'' The
transferred proteins were probed with anti-ASGP-R antibody and detected
with
I-protein A. Autoradiograms of the samples are shown
on the left with the corresponding densitometric scans on the right.
We next examined surface ligand and antibody binding in intact isolated hepatocytes at 4 °C to determine if alterations in surface receptor content followed a similar pattern as was observed for total receptor content. Results of these studies showed that both ASOR and antibody binding to surface receptors were significantly impaired after all three periods of ethanol exposure (Fig. 3). The impairments in ASOR binding were similar after all three time periods, while antibody binding was decreased to a greater extent after the longer 5-week period. These results indicated that after the early 4-day period, surface receptors were internalized but may have been unable to recycle back to the plasma membrane, since surface antibody binding was decreased without a corresponding decrease in binding to the total receptor population. To more closely examine a possible difference in receptor distribution between cells of control and ethanol-fed animals, we expressed the antibody binding data as a ratio of surface binding to total binding after the three periods of ethanol feeding (Table 1). Surface receptors accounted for 53% of the total population of receptors in control animals for the 4-day and 1-2-week feeding periods. In contrast, in the ethanol animals, only 39% of the total receptor number was found on the surface of the cells. These results indicate an intracellular accumulation of receptors in the ethanol animals after the early and intermediate times of feeding. After the chronic feeding period of 5 weeks, no differences in receptor distribution were observed between control and ethanol animals (Table 1).
Figure 3:
Determination of surface ASGP-R content
and activity over a time course of ethanol treatment. Animals were fed
liquid diets containing ethanol () as 36% of calories or an
isocaloric control diet (
) for 4 days, 1-2 weeks, and 5
weeks. The binding of
I- ASOR (A) and
anti-ASGP-R antibody and
I-protein A (B) to
intact isolated hepatocytes at 4 °C was performed as described
under ``Materials and Methods.'' Results are expressed as
femtomoles bound per million cells and are means ± S.E. for six
determinations. Values that are significantly different from controls
are indicated (*, p < 0.05).
We wanted to gain more information about the mechanism(s) responsible for these alterations in receptor activity and content that had occurred after ethanol feeding. For these studies, we chose to study an early feeding period of 6-8 days and a longer chronic period of alcohol administration of 5-7 weeks since these were the two periods that distinguished changes in receptor content and activity. Similar to the first series of experiments described above, ligand binding was significantly decreased after both 6-8 days and 5 weeks of feeding in this new series of feeding experiments. The decreased ligand binding after the early 6-8-day feeding period was again due to receptor inactivation, since antibody binding was not impaired after this period (data not shown). Antibody binding was, however, significantly decreased in the ethanol cells after the chronic 5-week feeding period. We then used this series of animals to examine the expression of the asialoglycoprotein receptor gene encoding the major component of the rat receptor (RHL-1) by Northern blot analysis. Fig. 4shows a typical Northern blot for the ASGP-R RNA that was extracted from the cells of animals fed for the 6-8-day and 5-week periods. The same blots were stripped and reprobed with glyceraldehyde-3-phosphate dehydrogenase, and the results normalized to this internal standard. When results of six to eight pairs of animals were analyzed, the mRNA level for the RHL-1 subunit of the ASGP-R was significantly decreased by 30-50% (p < 0.05) after 5 weeks of ethanol exposure; whereas after early treatment, no difference was seen in the ethanol-fed animals as compared to controls.
Figure 4: Northern blot analysis of ASGP-R mRNA in isolated hepatocytes after 1 and 5 weeks of ethanol administration. mRNA was obtained from animals fed a control diet or ethanol as 36% of calories for 1 and 5 weeks. The extracted mRNA was fractionated on agarose gels, transferred to nylon membranes, and hybridized with the random-labeled cDNA probes as described under ``Materials and Methods.'' The expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also shown, indicating uniform loading of the RNA samples onto the gel. The results shown are one representative Northern blot from six independent experiments.
To correlate the observed decreases in ASGP-R mRNA levels
with protein synthesis, biosynthetic labeling experiments were
performed. Hepatocytes from the 5-week chronically treated animals were
incubated with either 2 mM [H]leucine or
trace amounts of [
S]methionine for up to 90 min
at 37 °C in methionine or leucine-free media. The higher
concentration of leucine was used to expand the extracellular leucine
so that the specific activity of intracellular leucine was maintained
at the same level as that of extracellular leucine. Labeling with
[
S]methionine provided higher specific activity
for labeling. As shown in Fig. 5, the incorporation of either
amino acid into the total protein pool was not altered after the
chronic administration of ethanol. Larger aliquots were used to
determine the incorporation of [
S]methionine
into the newly synthesized ASGP-R. Data from a representative labeling
experiment examining radioactivity in purified ASGP-R is shown in Fig. 6and shows that ASGP-R production increased over the 90-min
time course of labeling in hepatocytes isolated from both the control
and ethanol-treated animals and that the bands are noticeably less
intense at each time point in the ethanol samples. When data from six
to eight pairs of animals were analyzed, the amount of
[
S]methionine incorporated into the ASGP-R was
significantly decreased by an average of 50% at all time points during
the incubation period in the ethanol-fed animals as compared to
controls. These results indicate that the reduced ASGP-R transcript
levels after prolonged ethanol ingestion lead to a reduction in the
synthesis of the receptor.
Figure 5:
Determination of total protein synthesis
from 2 mM [H]leucine and trace
[
S]methionine. Hepatocytes from 5-week
ethanol-fed (
) and control (
) rats were incubated with
either 2 mM [
H]leucine (panel
A) or 200 µCi/ml [
S]methionine (panel B). At the indicated times, total trichloroacetic
acid-precipitable radioactivity was determined as described under
``Materials and Methods.'' Results are expressed as means
± S.E. for six pairs of animals. The values were not
significantly different between control and ethanol animals at any time
point.
Figure 6:
Time
course of ASGP-R synthesis after 5 weeks of ethanol administration.
Hepatocytes from control and ethanol-fed animals were incubated with
[S]methionine (100 µCi/ml). At the indicated
times, 1-ml aliquots were removed, and the ASGP-R purified utilizing
lactose column chromatography and immunoprecipitation as described
under ``Materials and Methods.'' The immunoprecipitated
receptor was subjected to SDS-PAGE, transferred to nitrocellulose
membranes, and the radioactivity in the receptor band was quantitated
using phosphorimager analysis. Data are presented as phosphorimager
units in the receptor band over the time course for a representative
pair of animals obtained from six to eight experiments. Values
significantly different from controls are indicated (*, p <
0.05). Inset, A typical phosphorimager pattern for newly
synthesized receptor in 90-min control (C) and ethanol (E) samples along with a corresponding lane showing
immunoprecipitation with non-immune serum.
We have previously shown that chronic ethanol administration alters multiple aspects of the hepatic RME pathway(1, 2, 3) . One major finding that consistently occurs is a decrease in ligand binding after as early as 1 week of ethanol administration, which persists through at least 6-8 weeks of feeding. The decreased ligand binding appears to be due to different reasons during these early (1-2 weeks) and later (5-8 weeks) periods of feeding. After more chronic ethanol administration (greater than 5 weeks), decreased binding appears to be due to an actual decrease in receptor content, which is characterized by decreases in both ligand and antibody binding. In contrast, after the earlier times of feeding, decreased ligand binding is not associated with decreased protein content for the ASGP-R, showing receptor inactivation. Along with this early inactivation of the receptor, an intracellular accumulation of receptors occurs in the ethanol cells. After the prolonged 5-week feeding period, however, the decreased receptor content in ethanol cells is reflected by equal decreases in surface and intracellular receptor number.
We also examined in greater detail the mechanism(s) responsible for the decreased receptor content that was apparent after the more chronic ethanol feeding period. The levels of mRNA for the ASGP-R in the two cell types was determined after both 1 and 5 weeks of feeding. Gene expression of mRNA for the receptor was decreased after 5 weeks of feeding, but not after the early 1-week feeding period. These data for ASGP-R mRNA correlated well with the levels of receptor protein that were present at the two periods. When Zern and co-workers (23) examined the effect of ethanol administration on another liver-specific gene (that for albumin synthesis), they found that chronic ethanol ingestion actually stimulated albumin mRNA content in vivo in rats. An increase in mRNA for albumin was also noted in baboons after chronic ethanol feeding(24) . The results we present here show a decreased mRNA content for ASGP-R after 5 weeks of ethanol feeding. Since the ASGP-R is a protein that is synthesized in the liver, it appears that not all mRNAs of genes responsible for liver-specific protein production are equally affected after ethanol administration. Along with the decreased mRNA content for the ASGP-R, the ability of the cells from ethanol-fed animals to synthesize ASGP-R from labeled methionine was also impaired after the chronic ethanol feeding periods. In contrast to the decreased receptor synthesis, amino acid incorporation into the total cellular protein pool was not altered in cells from ethanol-fed animals. Work from other laboratories on the effect of ethanol administration on protein synthesis in vitro in hepatocytes, as well as in isolated microsomes from mice and rats, likewise did not find ethanol-induced alterations in incorporation of amino acids into the total protein pool (25, 26) . We do not know at this time whether defects in post-transcriptional processing by the mRNA for the ASGP-R are present in alcohol-fed animals; however, the fact that message level was decreased to a similar extent as was receptor synthesis would indicate that the decreased receptor mRNA levels alone could explain the decreased in vitro synthesis of the receptor.
The early effects of ethanol administration on receptor inactivation and redistribution were also intriguing and may be related to each other. The build-up of intracellular receptors resulting in altered distribution could be due to a variety of reasons, two of which could be very prominent. One mechanism would be an impaired ability of internalized surface receptors to recycle back to the plasma membrane for reuse. In support of this model, we have previously shown that recycling of the ASGP-R is impaired after ethanol feeding(1) . Receptor recycling is dependent on successful dissociation of receptor-ligand complexes inside the cell, and a decrease in the dissociation of receptor and ligand after ethanol feeding could contribute an additional mechanism, which would lead to an intracellular accumulation of receptors. Indeed, we have previously reported that one alteration in the RME process after ethanol administration is a decreased ability of the ligand, ASOR, to dissociate from its receptor during continuous endocytosis(2, 27) . During normal operation of the RME cycle, internalized receptor-ligand complexes are transported by way of endosomal vesicles, and dissociation occurs within these vesicular compartments. An acidic intravesicular pH in the endosomes is thought to play a major role in the dissociation of receptor-ligand complexes and aid in the subsequent transport and routing of both receptor and ligand. We have recently reported that there is a decreased ability of the endosomes from ethanol-fed animals to acidify in the presence of added ATP(28) . This impaired acidification of endosomes after ethanol treatment could result in alterations in dissociation of receptor and ligand and contribute to decreased receptor recycling and altered receptor function.
Weigel et al.(9) have postulated that, in addition to endosomal acidification, receptor-ligand dissociation may be influenced by factors that affect functioning of the receptor itself, such as receptor inactivation. This second explanation for altered dissociation is also an attractive one and one that may be relevant to our system of examining ethanol-induced effects of ligand processing and receptor function. The ASGP-R pool is composed of two classes, or subsets, of receptor types. One set of receptors, State 2 receptors, is responsible for the bulk of endocytosis activity by the receptor (>80%), having been shown to constitutively recycle (even in the absence of ligand) and to participate in a reversible inactivation/reactivation cycle during the recycling pathway(9) . The reversible inactivation/reactivation cycle is thought to facilitate both receptor-ligand dissociation and segregation of free receptor and ligand. Activity and recycling of these State 2 receptors are susceptible to modulation by a variety of agents, including azide (a metabolic energy poison), colchicine (an agent that disrupts microtubules), and monensin and chloroquine (lysosomotrophic agents). These agents can induce inactivation and/or redistribution of the State 2 receptors(7, 8) . Medh and Weigel (29) have shown that the inactivation/reactivation cycle for the ASGP-R can be reconstituted in permeabilized rat hepatocytes. In those studies, treatment with ATP showed a time-dependent and dose-dependent inactivation of the receptor. The fact that ATP produced inactivation suggests that the activation or inactivation of the receptor may be linked to the phosphorylation state of the receptor. Additional work from Weigel's laboratory also indicated that phosphorylation regulates ASGP-R function and/or receptor distribution within the cell(15) . In the present report, we show that ethanol administration results in receptor inactivation and redistribution to the interior of the cell early during the feeding period. The inactivation/redistribution of the ASGP-R after early ethanol administration, which we have identified is intriguing and may indicate that ethanol has an effect on the State 2 receptor population in liver cells. It will be interesting to examine whether these changes after ethanol administration are accompanied by changes in the phosphorylation state of the receptor.
In summary, we have shown in an animal model of ethanol administration that function of a liver-specific receptor, the ASGP-R, is altered. Early during the feeding scheme (up to 2 weeks), ligand binding to the receptor is decreased without a corresponding change in protein content. Upon prolonged feeding, decreased ligand binding is reflected by an actual decrease in protein content of the receptor. The decreased protein content is accompanied by a loss of ASGP-R-specific mRNA and a decreased ability of the receptor to be synthesized from labeled amino acids. The early changes in receptor function are characterized by a loss of ligand binding without a loss of protein or mRNA content. The ``inactivated'' receptor we have described here may be subject to degradation and/or altered processing once inside the cell. The mechanism and consequences of this inactivation must be assessed by further studies to determine its role in alcohol liver injury and the concomitant altered protein trafficking, which results during this injury process.