Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, Minnesota 55905
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ABSTRACT |
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Ethanol has been predicted to alter vesicle-based protein traffic in hepatocytes, in part, via a disruption of the microtubule (MT) cytoskeleton. However, information on the effects of chronic ethanol exposure on MT function in vivo is sparse. Therefore the goal of this study was to test for ethanol-induced changes in rat liver tubulin expression, assembly, and cellular organization, using molecular, biochemical and morphological methods. The results of this study showed that tubulin mRNA and protein levels were not altered by ethanol. Tubulin, isolated from control and ethanol-fed rats, showed similar polymerization characteristics as assessed by calculation of the critical concentration for assembly and morphological structure. In contrast, the total amount of assembly-competent tubulin was reduced in livers from ethanol-fed rats compared with control rats when assessed by quantitative immunoblot analysis using a tubulin antibody. In addition, we observed that MT regrowth and organization in cultured hepatocytes treated with cold and nocodazole was markedly impaired by chronic ethanol exposure. In summary, these results indicate that tubulin levels in liver are not reduced by ethanol exposure. While there is a substantial amount of tubulin protein capable of assembling into functional MTs in ethanol-damaged livers, a marked portion of this tubulin is polymerization incompetent. This may explain why these hepatocytes exhibit a reduced number of MTs with an altered organization.
tubulin; liver; nocodazole
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INTRODUCTION |
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HEPATOCYTES ARE KNOWN TO perform multiple secretory and endocytic functions. It has been well documented that the elaborate vesicle trafficking-transport machinery that supports these functions is markedly disrupted by ethanol exposure (3, 30). Several studies have demonstrated the effects of acute and chronic ethanol exposure on this process, using liver slices (28), isolated hepatocytes (17), intact liver (33), and human liver biopsies (19). All of these studies (17, 19, 28, 33) indicated that ethanol induces a striking intracellular retention of nascent proteins. This accumulation is believed to be responsible in part for the dramatic increase in diameter or "ballooning" of hepatocytes during the initial stages of ethanol-induced liver injury. It is also widely accepted that chronic ethanol exposure alters the ability of hepatocytes to endocytose and degrade proteins. Careful studies monitoring the endocytosis of a variety of ligands such as asialoglycoprotein (5) and epidermal growth factor (7) have shown that ethanol induces a marked decrease in the processing of these macromolecules.
The microtubule (MT) cytoskeleton is believed to sustain multiple vesicular-based transport events in the hepatocyte, including the secretion of albumin, low-density lipoproteins, and glycoproteins (9, 15), excretion of specific bile salts and lipids, the uptake and recycling of sinusoidal surface receptors, and the transport of lysosomes to the canalicular surface (34). It has not been specifically defined how MTs support these movements in the hepatocyte, although it has been shown that endosomes, secretory vesicles, transcytotic carriers, and the Golgi apparatus are associated with the MT-based molecular motors cytoplasmic dynein (11, 22) and kinesin. These vesicle-associated, mechanochemical enzymes hydrolyze Mg:ATP to translocate along the MT surface lattice (24, 32). Thus a well-organized MT cytoskeleton is essential for normal efficient vesicle transport in the hepatocyte. MTs are polymeric filaments composed of tubulin dimers that assemble and disassemble in a Ca2+- and GTP-sensitive manner from a centrosomal complex. In many cells this centrosomal nucleation site is situated about the nucleus at the cell center. The precise location of MT nucleation sites in the hepatocyte has been suggested to be at several distinct cytoplasmic sites such as perinuclear, pericanalicular (21), or the canalicular membrane itself (1).
Presently, the mechanisms by which MT assembly is regulated in
mammalian cells are complex and poorly defined. Because MT dynamics are
sensitive to minute changes in concentrations of divalent cations,
nucleotides, and exposure to drugs, it has been proposed that ethanol
or its reactive intermediate acetaldehyde might alter MT polymerization
and organization in hepatocytes. As demonstrated by Tuma and co-workers
(13, 14, 26, 28) and Volentine (33), ethanol oxidized to acetaldehyde
forms both stable and unstable adducts with the free -amino group of
lysyl residues of
-tubulin. Substoichiometric
concentrations of acetaldehyde were shown to significantly inhibit
polymerization into functional MTs (4, 18, 26). Although the
concentrations of acetaldehyde used in these in vitro studies were low
(10-100 µM), it has not yet been clearly defined if ethanol
and/or acetaldehyde alters MT polymerization and organization
in the living hepatocyte. Previous in vivo studies (3, 18) of tubulin
in ethanol-damaged rat livers have suggested that there is a reduction
in the amount of polymerized tubulin and morphological alterations in
MT structure. In contrast, however, other similar studies (4) have
shown no difference in MT number or structure.
In this study we have combined both biochemical and morphological methods to define the effects of chronic ethanol exposure on MT polymerization and organization in hepatocytes from the rat liver. Northern and Western blot analyses, as well as immunofluorescence microscopy, indicate that levels of tubulin mRNA and protein in ethanol-damaged livers are the same as in control livers. Initial biochemical and electron microscopic characterization of tubulin assembly kinetics also shows little difference between ethanol-damaged and control cells. However, studies using sensitive Western blot analysis of MT pellets and supernatants with antibodies to tubulin do show a marked decrease in the amount of polymerizable tubulin from ethanol-damaged rat livers. Furthermore, tubulin-immunofluorescence staining of cultured ethanol-damaged hepatocytes that were first treated with cold and nocodazole and then rinsed with warm drug-free medium to allow recovery shows a markedly impaired capacity to regrow MTs from the canaliculus compared with control cells. Taken together, these studies show that there is a substantial portion of tubulin in hepatocytes that is resistant to chronic ethanol exposure. In addition to this tubulin, there is a smaller yet significant amount of tubulin that is "ethanol sensitive," and assembly incompetent as originally suggested by Baraona et al. (3). The implications of ethanol perturbation on MT assembly and vesicle transport are discussed.
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MATERIALS AND METHODS |
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Animal treatment. Male Sprague-Dawley rats (150-200 g) were purchased from Harlan Sprague-Dawley (Cincinnati, OH). Rats were paired according to weight and were fed a Lieber-DeCarli diet (Bio-Serv, Frenchtown, NJ) containing 36% of calories from either ethanol or isocaloric carbohydrate (2) for 8-17 wk. All rats were fed daily at 4:00 PM, although completion of the liquid meal could take anywhere from 2 to 6 h. These rats were killed in the morning between 8:00 and 9:00 AM. Thus the rat livers were utilized 12-16 h after an ethanol insult.
Cell culture. Primary cultures of rat hepatocytes were prepared by the isolation method of Gores et al. (12). Briefly, rats were anesthetized, and isolated livers were perfused via the portal vein with Ca2+- and Mg2+-free HEPES buffer. Subsequent perfusion included 1.0 mM Ca2+ and 0.02% collagenase. The livers were then gently raked, the cell suspension was centrifuged, and the resulting cell pellet was resuspended in DMEM containing the following additives: 0.1% BSA, 200 U/ml penicillin, and 200 mg/ml streptomycin. The cells were plated at a density of 5 × 105 cells on rat tail collagen-coated glass coverslips. After 3-5 h in culture, the cells were either fixed for immunofluorescence studies of MTs, or disassembly of MTs was induced with nocodazole (see immunofluorescence experiments described below).
Northern blot analysis.
Total RNA was extracted by the guanidinium thiocyanate method (6) from
the livers of control and alcohol-fed rats. RNA was separated on 1%
formaldehyde-agarose gels, transferred onto Hybond-N membrane
(Amersham, Arlington Heights, IL), and cross-linked for 12 s in an
ultraviolet cross-linker (Fisher). Membranes were prehybridized for 4 h
at 68°C with RapidHyb (Amersham) containing denatured salmon sperm
DNA at 100 µg/ml. Hybridization was done overnight under the same
conditions with random-primed
32P-labeled cDNA probes at 3 ng/ml. Filters were washed in 2× SSC (1× SSC is 0.15 M NaCl
and 0.015 M sodium citrate, pH 7.0) and 0.1% SDS twice for 30 min each
time, then in 0.1× SSC and 0.1% SDS twice for 30 min each time
at 65°C. The filters were exposed to Kodak X-Omat AR
films at 80°C using intensifying screens. The autoradiograms
were scanned with a Umax scanner, and band density was analyzed using
the NIH Image program version 1.47. The data representing band
densities were normalized to the density of the band representing the
housekeeping gene GAPDH.
The probes for
-tubulin and GAPDH
were prepared previously by RT-PCR using specific primers (29).
Purification of rat liver tubulin. Rat liver MTs were prepared by a modification of the temperature-dependent MT assembly and disassembly procedure, as described previously (10). Livers from ethanol-fed and pair-fed control rats (8-12 wk) were homogenized in ice-cold PEM buffer (100 mM PIPES, pH 6.9, 1 mM EGTA, and 1 mM MgSO4) containing protease inhibitors (phenylmethylsulfonyl fluoride, leupeptin, and soybean trypsin inhibitor) and 0.1 mM GTP. Homogenates were then spun at 4°C sequentially at 16,000 g for 20 min, at 105,000 g for 30 min, and at 112,000 g for 30 min to obtain a clear supernatant. The clear protein solution was dialyzed against 8 M glycerol in PEM for 90 min at 4°C, then supplemented with GTP to 1 mM, nuclease to 30 µg/ml, and protease inhibitors and incubated at 37°C for 30 min to polymerize MTs. The polymerized MTs were spun down at 105,000 g for 30 min at 30°C, and the supernatant (HS1) and MT pellets (HP1) were obtained. HP1 was resuspended in PEM containing protease inhibitors and incubated on ice for 30 min to disassemble MTs. The cold solubilized tubulin was then centrifuged at 65,000 g for 30 min at 4°C. The resulting pellets (CP2) were discarded, and supernatants (CS2) were mixed with an equal volume of 8 M glycerol in PEM and incubated at 37°C to induce the second MT assembly. To induce MT assembly from HS1, which contains unassembled tubulin in ethanol-damaged livers (see Fig. 5B), Taxol was added to HS1 to a final concentration of 20 µM and the mixture was incubated for 20 min at 37°C. The polymers were sedimented by centrifuging at 105,000 g for 30 min.
Measurement of tubulin critical concentration for assembly. Critical concentration (Cc) for MT assembly was obtained by MT pelleting assays. MTs were prepared from livers of ethanol-fed and control rats by two cycles of MT assembly and disassembly as described above. Serial dilutions of tubulin were performed followed by one cycle of assembly (37°C) and disassembly (4°C) of MTs. The concentration of cold-reversible MT polymer in the supernatant reflects the proportion of the tubulin that assembles into MTs. For the determination of Cc, the concentration of cold-reversible MT polymer rather than the concentration of total polymer (centrifuged pellet after MT polymerization) was used to eliminate inaccuracies due to the presence of denatured tubulin in the polymer pellet. The concentration of polymerized MTs was then plotted as the function of initial tubulin concentration in the corresponding tubes and visualized as a line graph. The Cc at which MTs start to polymerize is obtained by reading the values at the interception on the x-axis.
Western blot analysis and calculation of polymerized vs. soluble
tubulin.
Proteins separated by SDS-PAGE were transferred to nitrocellulose
filters that were washed in PBS, blocked with 10% dry milk (wt/vol) in
PBS, and incubated with a primary monoclonal antibody to -tubulin
(Amersham). Proteins were visualized by chemiluminescent detection of
horseradish peroxidase-tagged secondary antibodies. Total tubulin in
control and ethanol-damaged livers was calculated via densitometric
comparisons of isolated liver tubulin to serial dilutions of purified
bovine brain tubulin of a known concentration.
Transmission electron microscopy of negative-stained rat liver MTs. MTs were prepared as described above, then diluted to 0.1-0.2 mg/ml concentration with assembly buffer (0.1 M PIPES buffer, 1 mM EGTA, 1 mM MgSO4, 1 mM GTP, and 5% glycerol). The samples were then pelleted, incubated with 20 µM Taxol for 30 min at 37°C, then fixed with 1% glutaraldehyde for 30 min. MTs were adsorbed onto carbon-coated Formvar films on slotted grids for 60 s, washed in PEM buffer, negatively stained with 1% uranyl acetate for 30 s, dried, and then observed with a JEOL 1200 transmission electron microscope.
Immunofluorescence studies of MTs in cultured hepatocytes. To stain MTs in cultured hepatocytes, isolated hepatocytes were quickly washed in MT stabilizing buffer (MTSB) (0.1 M PIPES, pH 6.95, 1 mM EGTA, 3 mM MgSO4, and 5% glycerol) and then permeabilized in 0.4% digitonin in MTSB for 6 min at 37°C. Cells were then fixed in 3.0% formaldehyde with 0.075% glutaraldehyde for 20 min at 37°C, washed three times quickly in Dulbecco's PBS (DPBS) at room temperature, quenched for 15 min in 0.01 M glycine, and washed twice in DPBS. Cells were incubated overnight at 4°C with primary antibody diluted in blocking buffer (5% goat serum, 5% glycerol, and 0.04% sodium azide in DPBS). After three 10-min washes in DPBS, cells were incubated with FITC-conjugated rabbit anti-mouse IgG for 60 min at room temperature, after which they were washed three times for 10 min each in DPBS and once in H2O, blotted dry, and mounted in Slowfade (Molecular Probes, Eugene, OR). Cells were viewed using a ×100 objective on a Zeiss Axiovert 35 epifluorescence microscope (Carl Zeiss). Images were photographed on Tmax p3200 with a 35-mm camera. For experiments that examined and compared MT reassembly in cells from control vs. ethanol-damaged livers, cells isolated in culture for 8-10 h were placed in 4°C culture medium containing 1-5 µM nocodazole for 30 min, then allowed to warm to 37°C for 30 min while in the presence of the drug. Next, cells were allowed to recover and regrow MTs by rinsing in drug-free medium for 0, 1, 5, 10, or 30 min before permeabilization, fixation, and immunostaining as described above.
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RESULTS |
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Because ethanol has been predicted to alter MT function (18, 26), we tested the effects of chronic ethanol exposure on tubulin expression, polymerization, morphology, and cytoplasmic organization using various techniques.
Levels of rat liver tubulin mRNA and protein appear unaltered by
ethanol.
To test if the levels of tubulin expressed in ethanol-damaged rat
livers differ from those of control livers, we first performed Northern
blot analysis to assess the steady-state levels of tubulin transcripts.
Total RNA was isolated from livers of rats fed the Lieber-DeCarli diet
either with or without added ethanol for 8 and 14 wk. RNA
was blotted to membrane, and the blots were probed with a cDNA fragment
encoding -tubulin, the autoradiograms were then scanned, and the
density of bands was normalized to the levels of housekeeping gene
(GAPDH) expression. As shown in Fig.
1A, for
the two separate experiments conducted there was no significant change
in the levels of tubulin transcripts in the livers of ethanol-fed rats
compared with control rats at either time point. Next, we tested for
changes in the levels of synthesized tubulin protein by performing
Western blot analysis on supernatants of homogenates from livers of
pair-fed rats in three distinct experiments. Again, tubulin levels were
unchanged in ethanol-damaged livers compared with control livers at
both time points (Fig. 1B).
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Effects of chronic ethanol exposure on tubulin polymerization: in vitro studies. Because we were unable to detect any significant changes in tubulin expression in livers damaged by chronic ethanol exposure, we next tested the ability of tubulin, isolated from these livers, to assemble into normal MTs. To our knowledge, isolation of polymerized MTs from ethanol-damaged rat livers has not been conducted previously. This is largely because tubulin is not a prevalent protein in the liver compared with the brain and obtaining adequate liver tissue from pair-fed rats is expensive and laborious. For these reasons we first conducted a straightforward and semiquantitative comparison of the amounts of assembly-competent tubulin isolated from control vs. ethanol-damaged livers. As described in MATERIALS AND METHODS, MTs were prepared from liver homogenates by warm-cold, assembly-disassembly cycling with special care to maintain equal volumes of material. Generally, the enrichment of tubulin is doubled with each warm-cold pelleting cycle. Equal volumes of cold supernatants (depolymerized tubulin) and warm pellets (polymerized MTs) from ethanol-damaged and control livers were separated on SDS-PAGE (Fig. 2), stained with Coomassie blue, and compared by scanning densitometry. In the three times this experiment was done, we observed no difference in the amount of polymerized tubulin that pelleted from control and ethanol-damaged livers.
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Ethanol-induced changes in MT polymerization and organization in
living hepatocytes.
While the biochemical data depicted in Fig.
5A suggest that tubulin polymerization
is reduced in ethanol-damaged liver, it is unclear whether this
reduction translates into a functional reduction within the living
hepatocyte. To test this, indirect immunofluorescence staining of
tubulin was performed on polarized hepatocyte couplets isolated in
culture from control and ethanol-damaged livers. As shown in Fig.
6, hepatocytes isolated from both livers show similar, if not identical, staining patterns, with many MTs extending outward from the canaliculus to the cell periphery in a
linear fashion. These indistinguishable patterns were seen
in over 100 cells examined that were isolated from two distinct pairs of livers. Thus by this method any potential changes in the number, length, and organization of MTs in hepatocytes exposed to ethanol were
not obvious. To extend these studies, we next compared the capacity of
MTs to repolymerize in cultured hepatocytes that were first treated
with cold temperatures and the MT poison nocodazole (see
MATERIALS AND METHODS). These
experiments enabled us to test for ethanol-induced changes in the
kinetics of MT assembly within the context of the cell while reducing
the number of total MTs to facilitate morphological viewing. After MT
disassembly in cells cultured from control and ethanol-damaged livers,
cells were rinsed in warm, drug-free medium for various recovery times
(5-60 min), then fixed and stained with tubulin antibodies
(MATERIALS AND METHODS). As shown in
Fig. 7, control hepatocytes fixed after 10 min of recovery in warm, drug-free medium show numerous long MTs
extending from the canaliculus into the peripheral
cytoplasm. The number of MTs at this early recovery time
point is reduced in comparison to the untreated cells shown previously
in Fig. 6, making it easier to resolve the nucleation and organization of these MTs. In comparison, those cells isolated from ethanol-damaged livers and also allowed to recover for 10 min show a greatly reduced number of MTs in the cytoplasm, with most tubulin staining restricted to the canalicular membrane. As cells from control livers are allowed
to recover from cold and nocodazole treatment over longer periods of
time (30 min), an increasing number of MTs polymerized from both the
canaliculus and discrete cytoplasmic foci. In ethanol-damaged cells,
even after 30 min recovery, MTs had still not assembled in large number
from the canaliculus. Instead, most assembled from cytoplasmic foci to
form large, bright asters.