ARTICLE |
Correspondence to: Lukas Landmann, Dept. of Anatomy, Pestalozzistr. 20, CH-4056 Basel, Switzerland.
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Summary |
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Hepatocellular Na+,K+-ATPase is an important driving force for bile secretion and has been localized to the basolateral plasma membrane domain. Cholestasis or impaired bile flow is known to modulate the expression, domain specificity, and activity of various transport systems involved in bile secretion. This study examined Na+,K+-ATPase after ethinylestradiol (EE) treatment and after bile duct ligation (BDL), two rat models of cholestasis. It applied quantitative immunoblotting, biochemical and cytochemical determination of enzyme activity, and immunocytochemistry to the same livers. The data showed a good correlation among the results of the different methods. Neither EE nor BDL induced alterations in the subcellular distribution of Na+,K+-ATPase, which was found in the basolateral but not in the canalicular (apical) plasma membrane domain. Protein expression and enzyme activity showed a small (~10%) decrease after EE treatment and a similar increase after BDL. These modest changes could not be detected by immunofluorescence, immuno EM, or cytochemistry. The data, therefore, demonstrate that Na+,K+-ATPase is only slightly affected by EE and BDL. They suggest that other components of the bile secretory apparatus that take effect downstream of the primary basolateral driving force may play a more prominent role in the pathogenesis of cholestasis. (J Histochem Cytochem 46:405410, 1998)
Key Words: Na+,K+-ATPase, rat liver, cholestasis, ethinylestradiol, bile duct ligation, Western blotting, enzyme activity, cytochemistry, immunocytochemistry, immuno EM
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Introduction |
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In most animal cells the major ion-translocating ATPase is the sodium pump or Na+,K+-ATPase (EC 3.6.1.3.). This plasma membrane enzyme maintains sodium and potassium gradients that are crucial for many physiological cell functions. Na+,K+-ATPase is a heterodimer consisting of a catalytic - and a glycosylated ß-subunit, which are expressed as several isoforms in a tissue- or cell-specific manner (
1- and ß1-isoform types (
In hepatocytes, a multitude of transport systems in the plasma membrane are involved in the regulation of intracellular ion homeostasis, membrane potential, intracellular pH, and ion transport (
Whereas functionally active Na+,K+-ATPase is localized in the basolateral domain of most polarized epithelial cells, controversial immunochemical data have been reported on the distribution of the holoenzyme and its subunits in hepatocytes. Only recently has a consensus emerged that functionally competent /ß-heterodimers are expressed in the basolateral plasma membrane domain only (
Cholestasis is a term for pathophysiological conditions that are characterized by reduced bile flow. Cholestasis has been shown to be associated with altered expression and/or activity of various transport systems (
Therefore, this study examined the expression, activity, and distribution of Na+,K+-ATPase in two animal models of cholestasis; EE for intrahepatic and BDL for extrahepatic obstructive cholestasis. We applied quantitative immunoblotting, biochemical and cytochemical determination of enzyme activity, and immunocytochemistry to the same rat livers. The data demonstrate that neither EE nor BDL induces alterations in the subcellular distribution of Na+,K+-ATPase, and that protein mass and enzyme activity are only slightly altered.
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Materials and Methods |
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Reagents
17-Ethinylestradiol was purchased from Sigma Chemical (St Louis, MO), pentobarbital (Nembutal) from Abbot Laboratories (North Chicago, IL), 1,2-propanediol from Fluka AG (Buchs, Switzerland), and the kit for determination of bile acids from Becton Dickinson Diagnostics (Orangeburg, NY). All other reagents were of the highest purity available from either Merck (Darmstadt, Germany), Fluka, or Sigma.
Animals and Treatments
Cholestasis was induced in male SpragueDawley rats (240275 g; BRL, Füllinsdorf, Switzerland) as described (
Cholestatic Parameters.
The degree of cholestasis was monitored as described (
Preparation of Livers
Animals were anesthetized and livers flushed free of blood as described (
Western Blot Analysis
To concentrate the homogenate, aliquots were centrifuged at 100,000 x g for 1 hr and resuspended in the buffer used for homogenization. After protein determination, equal amounts of protein of this total particulate fraction were separated by SDS-PAGE and further processed by Western blotting (1-subunit of Na+,K+-ATPase (Upstate Biotechnology; Lake Placid, NY) at a dilution of 1:1000 and antibody was visualized with iodinated protein A. In preliminary experiments, linear responses to the amount of protein and to various exposure times of the film were established (data not shown). The autoradiographs were densitometrically quantitated with a CAMAG TLC scanner II (CAMAG AG; Muttenz, Switzerland).
Determination of Enzyme Activities in Homogenates
Immediately after removal the right caudal and lateral lobes were homogenized (20% w/v) as described (
Cytochemistry
Fifty-µm tissue slices were collected in 100 mM Pipes buffer, pH 7.4. Na+,K+-ATPase activity was shown using the cerium technique, as described (
Immunofluorescence
Cryosections of 0.51 µm were reacted for indirect immu-nofluorescence as described (
Immunoelectron Microscopy
Ultrathin cryosections were picked up with 2.3 M sucrose and stored on drops of PBS containing 0.5% (w/v) BSA and 0.1% (w/v) gelatin (incubation buffer). The grids were floated at RT sequentially on drops of incubation buffer containing (a) 0.02 M glycine for 10 min, (b) 5% (v/v) goat serum for 15 min, and (c) the primary antibody IEC 1/48 diluted 1:100 (v/v) for 30 min. After washing, grids were transferred for 20 min onto drops of goat anti-mouse IgG-coated 10-nm colloidal gold (Amersham International; Amersham, UK) diluted 1:40 (v/v) in incubation buffer. Controls were floated on incubation buffer without antibody IEC 1/48. After washing, grids were floated on 2% (w/v) aqueous OsO4 for 15 min, washed three times with distilled water, floated on 0.5% (w/v) uranylacetate, and thin-embedded with 2% (w/v) polyvinyl alcohol (
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Results |
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Protein Expression
We first examined the expression of Na+,K+-ATPase at the protein level in control and cholestatic rat liver by Western blot analysis of the total particulate fraction. The data demonstrated that in all control and experimental groups the antiserum recognized a protein of identical electrophoretic mobility (Mr 110,000) corresponding to the 1-subunit (data not shown). To assess whether the amount of protein expressed was altered by cholestasis, we analyzed the immunoblots of all animals densitometrically. The data demonstrated wide differences among animals and indicated a chole-stasis-associated decrease by 11% (5223 vs 5856 in controls) after EE treatment and an increase by 15% (5433 vs 4709) in the BDL group.
Enzyme Activity in Homogenates
Enzyme activity decreased to 90% of the control level in the EE group (63.4 vs 70.3 mU/mg protein) and rose to 110% (52.9 vs 48.3) in BDL. These alterations are means of widely differing individual values. They are in line with our data on protein expression. However, the activity of aminopeptidase N, a canalicular plasma membrane enzyme that is not affected by BDL (
Cytochemistry
Na+,K+-ATPase activity in situ was demonstrated ultrastructurally with the cerium method (Figure 1, left column). Livers of control animals treated with propanediol alone (Figure 1A) or subjected to sham operation displayed enzyme activity at the basolateral plasma membrane of hepatocytes, whereas no reaction product was observed at the canalicular cell pole. No differences were found within the liver acinus. Neither distribution nor intensity of reaction product were altered by treatment with EE (Figure 1B) or BDL (Figure 1C). Rarely, the canalicular cell pole also displayed reactivity after BDL (Figure 1C, arrow).
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Immunocytochemistry
The subcellular distribution of Na+,K+-ATPase was examined morphologically by indirect immunostaining at the light and electron microscopic levels. Immunofluorescent localization of Na+,K+-ATPase in semithin sections (Figure 1, right column) showed that in control liver (Figure 1D) immunoreactivity was localized at the basolateral domain of hepatocytes and cholangiocytes but was absent in the canalicular plasma membrane. Neither EE (Figure 1E) nor BDL (Figure 1F) induced any appreciable alterations in the distribution pattern or in the intensity of immunofluorescence. Only BDL livers occasionally displayed a few weakly immunopositive canaliculi (Figure 1F, arrow).
The ultrastructural localization of Na+,K+-ATPase was demonstrated by immunogold labeling of ultrathin cryosections (Figure 2). In all animal groups the antigen was expressed exclusively in the basolateral plasma membrane (Figure 2, left), whereas the canalicular domain (Figure 2, right) was immunonegative. In addition, the density of gold label showed no appreciable alterations in the different groups. No intracellular membranes or vesicular structures showed immunoreactivity. Very few canaliculi in livers of the BDL group displayed any gold particles.
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Discussion |
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Cholestasis is associated with increased levels of cholephilic compounds in serum. This defect may be caused by paracellular regurgitation of bile constituents through defective tight junctions (
Our initial expectation of establishing a correlation between the three techniques at the animal level was not realized because no significant changes in Na+,K+-ATPase could be observed. Among the experimental groups, however, the results of the different methods agreed well. Our data indicate that protein expression and activity are not affected significantly by EE or BDL treatment. This parallels the subcellular protein distribution, which is not altered by either model of cholestasis.
A substantial decrease in EE-induced Na+,K+-ATPase activity has been reported previously (
Although most studies using cytochemical (-subunit is localized to the basolateral plasma membrane domain (
/ß-complexes are expressed in the basolateral membrane only.
BDL, but not EE, has been demonstrated to cause redistribution of domain-specific basolateral and canalicular plasma membrane proteins (
In conclusion, our data demonstrating unaltered subcellular distribution of Na+,K+-ATPase and only minor changes in protein expression, as well as in enzyme activity, suggest that this enzyme is only slightly affected by cholestasis. Therefore, other mechanisms appear to play a more decisive role in the pathogenesis of cholestasis.
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Acknowledgments |
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Supported by grants An 192/1-2 from Deutsche Forschungsgemeinschaft (SA), 34-40486.94 (LL), and 31-33520.92 (BS) from the Swiss National Science Foundation.
We thank J.P. Boeglin and R. Plank for skillful technical assistance, and Dr A. Quaroni for the kind gift of antibody IEC 1/48.
Received for publication April 16, 1997; accepted September 30, 1997.
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