Changes in G protein expression account for impaired modulation of hepatic cAMP formation after BDL

Bernard Bouscarel1,2, Yasushi Matsuzaki3, Man Le3, Thomas W. Gettys4, and Hans Fromm1

1 Division of Gastroenterology and Nutrition, Department of Medicine, and 2 Department of Biochemistry and Molecular Biology, The George Washington University Medical Center, Washington, District of Columbia 20037; 3 Division of Gastroenterology, Institute of Clinical Medicine, University of Tsukuba, Ibaraki 305, Japan; and 4 Division of Gastroenterology, Departments of Medicine and of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425-2223

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The regulation of cAMP synthesis by hormones and bile acids is altered in isolated hamster hepatocytes 2 days after bile duct ligation (BDL) [Y. Matsuzaki, B. Bouscarel, M. Le, S. Ceryak, T. W. Gettys, J. Shoda, and H. Fromm. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G164-G174, 1997]. Therefore, studies were undertaken to elucidate the mechanism(s) responsible for this impaired modulation of cAMP formation. Hepatocytes were isolated 48 h after either a sham operation or BDL. Both preparations were equally devoid of cholangiocyte contamination. Although the basal cAMP level was not affected after BDL, the ability of glucagon to maximally stimulate cAMP synthesis was decreased by ~40%. This decreased glucagon effect after BDL was not due to alteration of the total glucagon receptor expression. However, this effect was associated with a parallel 50% decreased expression of the small stimulatory G protein alpha -subunit (Gsalpha S). The expression of either the large subunit (Gsalpha L) or the common beta -subunit remained unchanged. The expression of Gialpha 2 and Gialpha 3 was also decreased by 25 and 46%, respectively, and was associated with the failure of ANG II to inhibit stimulated cAMP formation. Therefore, alterations of the expression of Gsalpha S and Gialpha are, at least in part, responsible for the attenuated hormonal regulation of cAMP synthesis. Because cAMP has been reported to stimulate both bile acid uptake and secretion, impairment of cAMP synthesis and bile acid uptake may represent an initial hepatocellular defense mechanism during cholestasis.

bile acid; ursodeoxycholic acid; adenosine 3',5'-cyclic monophosphate; isolated hamster hepatocytes; cholestasis

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

LIGATION OF THE GOLDEN SYRIAN hamster common bile duct represents a well-defined experimental model of extrahepatic cholestasis in humans (6, 23, 29). In this model, 2-day bile duct ligation (BDL) is associated with 20- and 30-fold increases in serum bilirubin and total bile acid concentration, respectively. These alterations occur in the absence of both hepatic necrosis and periportal or portal fibrosis (29).

cAMP is a second messenger known to be involved in hepatocellular regulatory processes, such as gluconeogenesis, glycogenolysis, and cell proliferation (11, 23). Recently, cAMP and its analogs have also been implicated in the stimulation of hepatocellular uptake and secretion of bile acids (1, 19, 25). Hormones, including vasoactive intestinal peptide and glucagon, promote hepatocellular cAMP synthesis from ATP, through the activation of adenylyl cyclase; this process is mediated by a stimulatory guanine nucleotide-binding protein (Gs). On the other hand, hormones such as ANG II inhibit adenylyl cyclase activity and cAMP synthesis mediated by an inhibitory guanine nucleotide-binding protein (Gi).

The membrane-associated G proteins exist as heterotrimers consisting of alpha - (40-52 kDa), beta - (35-36 kDa), and gamma -subunits (10 kDa) (18). The two Gsalpha isoforms with apparent molecular masses of 42-45 kDa (Gsalpha S) and 47-52 kDa (Gsalpha L) are produced by alternative splicing of Gsalpha mRNA. These Gsalpha isoforms have been identified in numerous tissues, including the liver (16, 42). The Gialpha proteins have been classified into three subtypes: Gialpha 1, Gialpha 2, and Gialpha 3 (see Ref. 17 for review). However, whereas the Gialpha 1 subtype is expressed in the brain and adipose tissue, only the Gialpha 2 and Gialpha 3 isoforms are detectable in the liver (16).

Sherwin et al. (38) and Schölmerich et al. (36) have observed hyperglucagonemia associated with a decreased gluconeogenic response to glucagon in cirrhotic patients and in cholestatic rats, respectively. Furthermore, the glucagon-induced cAMP formation has been found to be impaired after BDL, as measured both in rat hepatic membranes (35) and in isolated hamster hepatocytes (29). We and others have postulated that the cellular alterations of this hormonal responsiveness were due to a hepatic accumulation of bile acids during cholestasis (29, 36) and that these alterations were irreversible (29). However, the mechanism(s) responsible for the alteration of hormone-induced cAMP synthesis during cholestasis are still unclear.

Therefore, the aim of the present study was to gain information on the mechanism(s) responsible for the impaired modulation of cAMP formation in hamster hepatocytes isolated 2 days after BDL. The potentially irreversible alteration of the stimulatory mechanism of hormones, such as glucagon, as well as of the inhibitory mechanism of both ANG II and bile acids, in particular unconjugated ursodeoxycholic acid (UDCA) and its taurine conjugate tauroursodeoxycholic acid (TUDCA), was investigated in this hamster model of cholestasis. The regulation of adenylyl cyclase, expression of the G protein subunits, as well as the hepatocellular uptake of bile acids were the focus of the present study.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Surgical procedure, hepatocyte, and cholangiocyte isolation. Adult male Golden Syrian hamsters (100-130 g body wt) were maintained on a 12:12-h light-dark cycle and fed a standard rodent chow diet for 2 days after BDL or sham operation, as previously described (29). The hepatocytes were then isolated in parallel, i.e., from the livers of pairs of BDL and sham-operated animals, using the collagenase perfusion technique, as previously described (2-4, 29). Cholangiocytes were isolated according to the method described by Ishii et al. (22) from the remaining portal tract after isolation of the hepatocytes.

Determination of cellular cAMP levels and [14C]TCA uptake. The cAMP concentration was determined by RIA according to the method of Gettys et al. (15), as previously described (2, 29). The dose-dependent hepatocellular uptake of [14C]taurocholic acid (TCA) and [14C]UDCA, as well as the initial bile acid uptake rate, expressed as nanomoles per gram of cell per second, were determined by previously described methods (4).

Preparation of hepatocellular total and membrane fractions, as well as total fraction of HeLa cells and cholangiocytes. The total and membrane protein fractions of the hepatocytes were prepared as previously described (3). The isolated hepatocytes were incubated in a 20 mM Tris · HCl solution (pH 7.5), containing 250 mM sucrose, 10 mM EGTA, 2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml leupeptin, 0.1 mg/ml antipain, and 20 µM Triton X-114 for 20 min at 4°C. The cell homogenate was then centrifuged at 100,000 g for 30 min at 4°C. The pellet (membrane) fraction was further incubated with the same buffer containing 1% Triton X-100 for 30 min and centrifuged at 100,000 g for 30 min at 4°C, and the supernatant was retained as the membrane fraction. A similar method was used to isolate the total fraction of hepatocytes, HeLa cells, and cholangiocytes. Briefly, the respective cells were directly incubated with 1% Triton X-100 for 30 min at 4°C and processed according to the method described for the plasma membrane fraction. The total and membrane fractions were collected, concentrated using an Amicon C-30 filter (Beverly, MA), and stored at -70°C.

Immunologic detection of CK-19 and glucagon receptor. For the immunologic detection of cytokeratin-19 (CK-19), proteins (100 µg) from total HeLa cells, cholangiocytes, and total and plasma membrane hepatic fractions were separated by 8% SDS-PAGE, transferred to nitrocellulose membranes, and probed with a mouse monoclonal anti-CK-19 antibody (1:100) followed by a horseradish peroxide (HRP)-labeled rabbit anti-mouse IgG (1:750) secondary antibody. For the immunodetection of the glucagon receptor, 80 µg protein from the total hepatic fraction, as well as 3-6 µl of COS cell membrane fraction expressing the glucagon receptor (see Ref. 8 for details), were separated by 8% SDS-PAGE, transferred to nitrocellulose membranes, and probed with a rabbit polyclonal anti-glucagon receptor (ST-18) antibody (1:10,000) followed by an HRP-labeled donkey anti-rabbit IgG (1:4,000) secondary antibody.

Preparation of plasma membranes and immunologic detection of G proteins. Plasma membranes were purified from pooled livers of two to three BDL and sham-operated Golden Syrian hamsters according to the Percoll gradient technique of Prpic' et al. (32), as previously described (3). The plasma membranes were resuspended in 25 mM HEPES (pH 7.4) containing 140 mM NaCl, 100 µg/ml leupeptin, 1 µg/ml soybean trypsin inhibitor, and 1 mM EDTA, and stored at -70°C. For immunologic detection, the purified plasma membranes were solubilized on ice with 0.9% sodium cholate (pH 8.0) and the supernatant was collected after centrifugation at 13,000 g for 5 min at 4°C. The proteins were separated by SDS-PAGE (12.5% acrylamide, 0.051% N, N'-diallyltartardiamide) and transferred by electrophoresis to Immobilon-P polyvinylidine difluoride membranes (Millipore, Bedford, MA). After blocking, the polyvinylidine difluoride membranes were probed with an antiserum directed against the COOH-terminal decapeptide, residues 385-394 of Gsalpha S and Gsalpha L, residues 345-354 of Gialpha 2, residues 345-354 of Gialpha 3, or the peptide sequence residues 127-139 of common beta -subunits. The method of preparation of the different G protein antibodies used in the present study has been previously described by Raymond et al. (33). The detected proteins were visualized with 125I-labeled goat anti-rabbit IgG (1 × 106 counts · min-1 · ml-1) as described by Gettys et al. (16). Finally, the membranes were exposed overnight to Kodak XAR film with intensifying screens and analyzed by densitometric scanning.

Statistical analyses. The results are expressed as means ± SE. The statistical significance of differences among the means was determined by either the one-way ANOVA or the paired Student's t-test where appropriate.

Materials. UDCA and TUDCA were supplied by Tokyo Tanabe (Tokyo, Japan), and TCA was purchased from Steraloids (Wilton, NH). [24-14C]TCA (sp act 50-55 mCi/mmol) was purchased from NEN (Du Pont, Boston, MA). All bile acids used were 98-99% pure judged by either gas-liquid chromatography or HPLC. [24-14C]TUDCA (sp act 5.4-5.5 mCi/mmol) was a gift from Tokai Research (Daiichi Chemical, Ibaraki, Japan) and was 92.2% pure as judged by TLC. Sodium-125I was purchased from Dupont-NEN Radiochemicals (Boston, MA). Glucagon, ANG II, leupeptin, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor were from Sigma Chemical (St. Louis, MO). Forskolin was from Calbiochem (San Diego, CA). Other chemicals used were from Fisher (Pittsburgh, PA) and were of the highest purity available. The mouse monoclonal anti-CK-19 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-Gsalpha , -Gialpha 2, and -Gialpha 3 or common beta -subunit antibodies were a gift of Dr. T. W. Gettys. HRP-labeled rabbit anti-mouse and donkey anti-rabbit IgG were from Amersham (Arlington Heights, IL) and Miles Scientific (Neperville, IL). The rabbit anti-glucagon receptor (ST-18) antibody and the plasma membrane fraction of COS cells expressing the glucagon receptor were gifts of Dr. T. P. Sakmar, Rockefeller University (New York, NY).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of UDCA, TUDCA, and ANG II on glucagon-induced cAMP production in hepatocytes isolated from sham-operated and BDL hamsters. Although the basal cellular cAMP level was not significantly different from sham-operated hamsters (Table 1), the maximum glucagon-dependent cAMP formation was markedly decreased by ~50% in hepatocytes isolated from BDL hamsters (Table 1, Fig. 1, A and B). However, BDL did not compromise the glucagon potency (EC50) to stimulate cAMP synthesis (Table 1). Furthermore, the effect of 100 µM of either UDCA or TUDCA on the production of cAMP induced by increasing concentrations (0.01-100 nM) of glucagon was tested in hepatocytes isolated from both groups of hamsters (Fig. 1, A and B). In hepatocytes isolated from sham-operated hamsters, both UDCA and TUDCA inhibited the efficacy of glucagon with a maximum inhibition of ~40% (Fig. 1A), as previously reported (3, 29). ANG II also reduced the efficacy of glucagon in these hepatocytes (Fig. 1A). However, the inhibitory effect of UDCA and TUDCA was abolished in hepatocytes isolated from BDL hamsters (Fig. 1B). Furthermore, the lack of effect was not specific to bile acids, because ANG II also failed to decrease glucagon-induced cAMP production (Fig. 1B).

                              
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Table 1.   Glucagon stimulation of cAMP synthesis in hepatocytes isolated from sham-operated and BDL hamsters


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Fig. 1.   Effect of ursodeoxycholic acid (UDCA), tauroursodeoxycholic acid (TUDCA), and ANG II on production of cAMP induced by increasing concentrations (0.01-100 nM) of glucagon in hepatocytes isolated from sham-operated and bile duct ligated (BDL) hamsters. Hamster hepatocytes, isolated 2 days after either sham operation (A) or BDL (B), were incubated for 5 min with increasing concentrations of glucagon and in the presence and absence of 100 µM UDCA, 100 µM TUDCA, or 10 nM ANG II. At the end of this period the reaction was stopped by addition of 24% HClO4, and total cellular cAMP concentration was measured by RIA. Results are means of 3-4 experiments assayed in duplicate and are expressed as percentage of maximum cellular cAMP formation induced by 100 nM glucagon in hepatocytes from sham-operated hamsters (control, CTL). cAMP levels observed in the presence of glucagon concentrations >2.5 nM in hepatocytes isolated from BDL hamsters were significantly lower (B) than those in control hepatocytes (A; P < 0.05).

Expression of CK-19 and glucagon receptor in hepatocytes isolated from sham-operated and BDL hamsters. Previously, we have observed by light microscopy an increase in bile ductule cells (cholangiocytes) after BDL (29). Therefore, to rule out the possibility that contamination of the hepatocyte preparation with cholangiocytes could be responsible for the decreased glucagon-induced cAMP synthesis after BDL, we probed these cell preparations with an anti-cytokeratin-specific CK-19 antibody (8). We found (Fig. 2) that this antibody recognized a 43-kDa band in both HeLa cells and hamster cholangiocytes, whereas there was no significant detection in either total or plasma membrane fraction of hepatocytes isolated from BDL and sham hamsters. Furthermore, we have previously reported that neither the maximum number of binding sites nor the affinity of the glucagon receptor was significantly different in liver membranes isolated from BDL compared with sham hamsters (29). To confirm these results we studied the total expression of the glucagon receptor in hepatocytes from BDL and sham hamsters. The glucagon receptor expressed in COS-1 cells (8) was used as control. The analysis of the immunoblot shown in Fig. 3 indicated that the COS-1 proteins of 35, 55-75, and 110 kDa recognized by the ST-18 antibody were similar to results previously reported by Carruthers et al. (8). In addition, it was observed that this antibody recognizes the hamster hepatic glucagon receptor as a monomeric form of 68 kDa. Finally, there was no significant difference in the total expression of the glucagon receptor between sham and BDL hamsters.


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Fig. 2.   Comparative expression of cytokeratin (CK-19) in HeLa cells, hamster cholangiocytes, and hepatocytes. Proteins (100 µg) from total cellular fraction of HeLa cells and hamster cholangiocytes (Chol), as well as sham and BDL hamster hepatocyte membrane (M) and total fraction (T), were separated and immunoblotted as previously described in Refs. 3 and 29 and in MATERIALS AND METHODS, using a primary anti-CK-19 antibody and horseradish peroxide (HRP)-labeled secondary antibody. Immunoreactive proteins were analyzed as described by densitometric scanning (29) using photoimaging (Molecular Dynamics, Sunnyvale, CA). Results are representative of 3 experiments. * Significantly different from both HeLa cells and cholangiocytes, P < 0.05.


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Fig. 3.   Expression of glucagon receptor in hepatocytes isolated from sham-operated and BDL hamsters. Proteins from total cellular fraction of hamster hepatocytes and COS-1 cells were separated and immunoblotted as previously described in Refs. 3 and 29 and in MATERIALS AND METHODS, using primary antibody directed against rat glucagon receptor and HRP-labeled secondary antibody. Immunoreactive proteins were analyzed as described by densitometric scanning (29) using photoimaging (Molecular Dynamics). Results are representative of 3 experiments.

Dose-dependent effect of ANG II on forskolin-induced cAMP synthesis in hepatocytes isolated from sham-operated and BDL hamsters. To investigate the mechanism responsible for the decreased efficacy of glucagon to stimulate cAMP formation in hepatocytes isolated from BDL hamsters, the direct stimulation of adenylyl cyclase by forskolin was tested. The results are shown in Fig. 4. The production of cAMP induced by forskolin concentrations >10 µM was significantly decreased (P < 0.05) by 30-35% after BDL. Furthermore, 100 nM ANG II inhibited the production of cAMP induced by 10 and 100 µM forskolin by 60-65%, respectively, in hepatocytes isolated from sham-operated hamsters. However, the same concentration of ANG II inhibited the production of cAMP induced by 100 µM forskolin by <20% in hepatocytes isolated from BDL hamsters.


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Fig. 4.   Effect of 100 nM ANG II on production of cAMP induced by increasing concentrations (1-100 µM) of forskolin in hepatocytes isolated from sham-operated and BDL hamsters. For experimental details, see legend for Fig. 1 and MATERIALS AND METHODS. Hepatocytes isolated from sham-operated or BDL hamsters were incubated for 5 min with increasing concentrations (1-100 µM) of forskolin and in the presence of either 0.9% NaCl as control or 100 nM ANG II, and cellular cAMP content was determined. Each bar is representative of 3 separate experiments performed in triplicate. Results are expressed as femtomoles per milligram of cell wet weight. * Significantly different from respective control, P < 0.05. + Significantly different between sham-operated and BDL, P < 0.05.

Characterization of the expression of Gsalpha and Gbeta proteins in plasma membranes of sham and BDL hamster livers. An alternative explanation for the decreased efficacy of glucagon-induced cAMP formation in hepatocytes isolated from BDL hamsters involves an altered expression of the Gs. An antiserum directed against the COOH-terminal decapeptide of Gsalpha was used to compare expression in liver plasma membranes from sham-operated and BDL hamsters. Initial experiments were conducted to establish that equivalent amounts of protein from the plasma membranes of both sham and BDL hamsters were loaded on the gels for each experimental replicate. As shown in Fig. 5, not only was the same amount of protein loaded, as determined by Coomassie blue staining, but also the protein pattern was similar between the plasma membrane preparations from sham-operated and BDL hamsters.


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Fig. 5.   Coomassie blue-stained gels of solubilized plasma membrane proteins from sham-operated and BDL hamster livers. Hepatic plasma membrane fractions were partially purified from both sham-operated and BDL hamsters, as previously described by Prpic' et al. (32) and in MATERIALS AND METHODS. Equal amounts (25 µg/lane) of solubilized membrane protein were loaded onto the gel and separated by SDS-PAGE. Resolved proteins were visualized by Coomassie blue staining.

Furthermore, the results reported in Fig. 6A indicate that the 43- (Gsalpha S) and 48-kDa (Gsalpha L) forms of Gsalpha are differently expressed after BDL. In contrast, the 36-kDa common beta -subunit was expressed at comparable levels and was unaltered by BDL (Fig. 6B). Although the expression of the large 48-kDa form (Gsalpha L) was not significantly changed, the small 43-kDa form (Gsalpha S) was decreased by ~40-50% after BDL (Fig. 6C). The observation that the 48-kDa form of Gsalpha is not altered during BDL is consistent with observations made by Dixon et al. (10).


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Fig. 6.   Expression of stimulatory GTP-binding protein (Gs) alpha - and beta -common subunits in plasma membranes of sham-operated and BDL hamster liver. Equal amounts of protein (25 µg/lane) from sham-operated or BDL hamster liver were separated by SDS-PAGE and transferred to polyvinylidine difluoride membranes in a semi-dry blotter. Blots were probed with an antiserum directed against the COOH-terminal decapeptide of Gsalpha S and Gsalpha L (A) or peptide sequence (residues 127-139) of beta -common subunits (B), and detected proteins were visualized using 125I-labeled goat anti-rabbit IgG (1 × 106 counts · min-1 · ml-1). Autoradiograms were developed after overnight exposure with intensifying screens. Autoradiographs were analyzed by scanning laser densitometry and expressed as function of densitometric scans of the Coomassie gels (C). Blots are representative of 3 experiments. * Significantly different from respective sham-operated control, P < 0.05. 

Characterization of Gialpha protein expression in plasma membranes of sham and BDL hamster livers. ANG II is known to inhibit hormone-induced cAMP formation through activation of Gi. The expression of both isoforms of the Gi family, Gialpha 1-2 (40 kDa) and Gialpha 3 (41 kDa), known to be present in rodent liver membranes was reduced after BDL (Fig. 7, A and B). As shown in Fig. 7C, the plasma membrane level of Gialpha 2 was significantly reduced by 25%, whereas Gialpha 3 was reduced by 46% in BDL hamster livers.


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Fig. 7.   Expression of inhibitory G protein (Gialpha 2 and Gialpha 3) subunits in plasma membranes of sham-operated and BDL hamster liver. For experimental details, see legend for Fig. 6 and MATERIALS AND METHODS. Western blots of liver membrane protein from sham-operated or BDL hamsters were probed with antiserum directed against Gialpha 2 (A) and Gialpha 3 (B), and detected proteins were visualized using 125I-labeled goat anti-rabbit IgG (1 × 106 counts · min-1 · ml-1). Autoradiographs were analyzed by scanning laser densitometry and expressed as function of densitometric scans of Coomassie gels (C). Blots are representative of 3 experiments. * Significantly different from respective sham-operated control, P < 0.05.

Dose-dependent uptake of TCA and TUDCA in hepatocytes isolated from sham and BDL hamsters. We have previously reported that Gi was not involved in the bile acid-induced inhibition of stimulated cAMP synthesis (3). Furthermore, the bile acid had to cross the plasma membrane to be effective in lowering cAMP formation (2). Therefore, one of the possible mechanisms responsible for the impaired inhibitory effect of bile acids on hormone-induced cAMP production in hepatocytes isolated from BDL hamsters could be related to an altered ability of the bile acids to cross the plasma membrane. To test this hypothesis, the dose-dependent uptake of [14C]TCA by hepatocytes isolated from both experimental groups was examined. Radiolabeled TCA was used because the hepatocellular transporter for TCA has been well characterized and is thought to transport most of the taurine-conjugated bile acids (20), as well as a significant portion of the unconjugated UDCA (4). The sodium-independent bile acid uptake was determined by incubating the cells under the same conditions as previously described, with the exception that sodium was replaced by choline. The sodium-dependent bile acid uptake was determined by subtracting the sodium-independent hepatocellular uptake from the total uptake. TCA was taken up by hepatocytes isolated from sham-operated hamsters in a dose-dependent manner, with a maximum uptake observed at TCA concentrations of ~20 µM (Fig. 8A). Under these conditions, >80% of the TCA uptake was sodium dependent. At a concentration of 100 µM, the sodium-independent uptake of TCA represented <25% of the total uptake (Fig. 8A). The sodium-dependent uptake of TCA was characterized by a maximal velocity of 2.9 ± 0.21 nmol · g of cells-1 · s-1 and a Michaelis constant of 24.8 ± 4.6 µM. In contrast, the total uptake of TCA by hepatocytes isolated from BDL hamsters did not reach saturation, was drastically reduced, and was mainly sodium independent (Fig. 8B). The sodium-independent TCA uptake was similar in hepatocytes from sham-operated hamsters with a rate of uptake of 0.012 nmol · g of cells-1 · s-1 · µM-1. In sham-operated hamsters, the sodium-dependent uptake of 100 µM [14C]TUDCA represented >80% of the total uptake with a maximal velocity and Michaelis constant of 2.2 ± 0.4 nmol · g of cells-1 · s-1 and 68 ± 24 µM, respectively. Similar to uptake of TCA, the sodium-dependent uptake of TUDCA was almost completely abolished in hepatocytes isolated from BDL hamsters.


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Fig. 8.   Dose-dependent uptake of [14C]taurocholic acid (TCA) by hepatocytes isolated from sham-operated or BDL hamsters. Hepatocytes isolated from sham-operated (A) or BDL (B) hamsters were incubated with increasing concentrations (1-100 µM) of TCA in bicarbonate buffer containing 145 mM sodium or in buffer in which sodium had been replaced by choline. At indicated period of time, duplicate aliquots were removed and centrifuged through an oil layer in centrifugation tubes as previously described (4). Sodium-dependent TCA uptake was determined by subtracting bile acid uptake in choline buffer (sodium independent) from that in the sodium-containing buffer (Total). Results are expressed as nanomoles per gram of cells per second and are representative of 3 different experiments performed in triplicate. SE values were <10% of the means and were omitted for clarity of presentation.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The results of the present study show that the modulation of cAMP formation by glucagon, ANG II, and bile acids is compromised in hepatocytes isolated from Golden Syrian hamsters 2 days after BDL. This decreased glucagon-induced cAMP formation after BDL is not due to alteration of the total expression of the glucagon receptor. These results are supportive of previous studies showing that the decreased efficacy of glucagon to stimulate cAMP formation was independent of any changes in either the affinity or number of glucagon receptors (29). Furthermore, this decreased glucagon-induced cAMP formation is not due to dilution of the cellular preparation with cholangiocytes during the isolation procedure of hepatocytes from BDL hamsters. Indeed, whereas immunochemical techniques allowed us to detect a protein band of ~43 kDa corresponding to CK-19, specific for both HeLa cells and cholangiocytes (22), this band could not be detected in either total or plasma membrane preparations of hepatocytes isolated from either BDL or sham-operated hamsters. These results suggest homogeneity of the hepatic parenchymal cell preparation under both experimental conditions.

One of the possible mechanisms responsible for the attenuated transmission of the hormonal signal from the glucagon receptor to the adenylyl cyclase could involve either an alteration or a decreased expression of Gs. The results of the present study indicate that the expression of Gsalpha L was either not changed or slightly increased, whereas that of Gsalpha S was decreased by 40-50% after BDL.

Recently, Yagami (42) reported that in liver membranes the beta -adrenergic receptor was only coupled to Gsalpha L, whereas the glucagon receptor was coupled to both Gsalpha L and Gsalpha S. Therefore, in agreement with these results, the 40-50% decrease in Gsalpha S expression observed in the present study can, to a certain extent, explain the decreased cAMP production induced by glucagon during cholestasis. Furthermore, the present results are also consistent with those of the study of Pecker et al. (31), which show that although no alteration in hepatic cAMP production was observed with the beta -adrenergic receptor agonist isoproterenol, a decreased glucagon-induced cAMP synthesis was observed in patients with liver cirrhosis.

In the present, as well as in a previous study, we have shown that the 40-50% decrease in glucagon-induced cAMP formation caused by certain bile acids was due to the bile acid-induced uncoupling of the glucagon receptor and Gs (3, 29). Furthermore, after BDL, glucagon is still able to stimulate cAMP synthesis, even if the maximum stimulatory effect of glucagon is only 50-60% of that of sham (Fig. 1 and Ref. 29). Because glucagon has been shown to activate both Gsalpha S and Gsalpha L, the stimulatory effect of glucagon on cAMP synthesis suggests a certain level of coupling of the glucagon receptor and Gs after BDL. Because the pool of Gsalpha S has been reduced but not abolished after BDL (Fig. 4, A-C), fewer Gsalpha S can be activated by glucagon, and therefore, UDCA are still able to uncouple certain of these receptors and Gsalpha S, but higher concentrations of the bile acid are required (29). The fact that after BDL bile acids are less able to decrease the hormone-induced cAMP production suggests that the mechanism altered after BDL is the same as that which is regulated by bile acids, and that the pool of Gs that is decreased after BDL is the major pool of Gs regulated by bile acids.

The altered expression of the G protein 2 days after BDL is not unique to Gs because the expression of Gi and the associated effect of ANG II are also altered. Use of hepatic plasma membrane preparations in several studies (14, 30) has suggested that Gi was exerting a "tonic" inhibitory action on adenylyl cyclase activity. Therefore, the BDL-associated reduced expression of Gi, as observed in our study, should have resulted in an increased basal cAMP level. However, as reported in Table 1, the basal cAMP level was not significantly altered after BDL. Thus this invalidates the concept of a tonic inhibitory action of Gi in our model. The present results are more supportive of those studies suggesting that increased cAMP levels are associated with increased expression or stimulation of Gs (27, 43).

The present findings suggest a decreased expression of both Gialpha 2 and Gialpha 3 by 25 and 46%, respectively, after BDL. The question remains whether the ANG II receptor is preferentially coupled to either or both of these subtypes (Gialpha 2 or Gialpha 3). However, the decrease of both Gialpha subtypes is compatible with the decreased inhibitory response observed with ANG II after cholestasis. Although not unanimously accepted, previous studies by Bushfield et al. (5) and by Remaury et al. (34) have suggested that Gialpha 2 rather than Gialpha 3 was responsible for the inhibition of the adenylyl cyclase. Gialpha 3, on the other hand, has been suggested to be involved in other cellular processes, including regulation of sodium transport (7). However, the fact that ANG II failed to inhibit glucagon-induced cAMP formation in BDL suggests one or more of the following events: 1) the 25% decrease in the Gialpha 2 protein during BDL could represent that Gialpha pool that possesses the major inhibitory action; 2) not only Gialpha 2 but also Gialpha 3 proteins are involved in the inhibition of adenylyl cyclase activity induced by ANG II; 3) whereas only 25% of the Gialpha 2 pool has disappeared, a large portion of the remaining Gialpha pool is not functional; and finally 4) an additional mechanism involved in the ANG II-induced inhibition of cAMP synthesis is impaired after BDL.

Furthermore, it is not possible at the present time to completely understand the discrepancy between the present results and those of the previous study by Rodriguez Henche et al. (35). In the latter study, the authors have reported no change in Gsalpha expression in hepatic membranes isolated from cholestatic rats. Furthermore, although both studies report the decreased expression of Gialpha 3 in hepatic membranes after BDL, in contrast to the present study, an increase of Gialpha 2 was reported in the rat model (35). Different hypotheses can be proposed to explain the discrepancy between these two studies. Dixon et al. (10) have reported differences in the distribution of the Gsalpha S and Gsalpha L proteins between the basolateral and apical hepatic membranes. Furthermore, Young et al. (43) have underlined the plasma membrane preparation as a potential source of differences in the results of studies on the hepatic regulation of adenylyl cyclase. Therefore, different techniques of liver membrane isolation could potentially lead to a different yield of isolated membranes and thus to different results. However, similar expression of the common beta -subunit between the two groups suggests similarities with our study as far as the yield and recovery of the liver plasma membrane fraction is concerned. However, it cannot be ruled out that the disparity observed in these two studies is due to the differences in the respective rodent model used.

The results of the present study also suggest that in addition to the G protein, the adenylyl cyclase could be affected during BDL-induced cholestasis. The direct stimulation of the cyclase by the nonhormonal diterpene forskolin was also significantly depressed during cholestasis. However, although forskolin is known to stimulate adenylyl cyclase directly (37), Insel et al. (21) have suggested that forskolin could be active through Gs or at least that Gs is required for full activation of adenylyl cyclase by forskolin. The latter hypothesis is in keeping with the findings in our study of a decreased expression of Gsalpha S during cholestasis. Nevertheless, it remains possible that the expression of adenylyl cyclase is also reduced during cholestasis.

The results of the present study are among the first to demonstrate that in hepatocytes isolated from BDL hamsters the sodium-dependent bile acid uptake mechanism is drastically decreased. These results are supported by those of Gartung et al. (13), which showed that in the rat model, in addition to the expressed protein, the mRNA for the hepatic bile acid transporter was decreased during cholestasis. Furthermore, although the sodium-independent bile acid uptake mechanism is not affected during BDL, unconjugated UDCA, chenodeoxycholic acid, and deoxycholic acid, which have been shown to cross the membrane of cells that do not possess a bile acid transporter (2), did not significantly inhibit stimulated cAMP synthesis (29). Therefore, this suggests that the decreased sodium-dependent bile acid uptake is not the major mechanism responsible for the reduced inhibitory effect of bile acids on stimulated cAMP synthesis.

It is worthwhile to mention that although BDL for 2 days should result in hepatic accumulation of bile acids, we have previously shown that there was no detectable amount of bile acid associated with the liver cells after the isolation procedure (29). This suggests therefore that any alteration of the glucagon-induced cAMP synthesis due to hepatic accumulation of bile acids had to have occurred before the liver cell isolation procedure. The persistence of this alteration in the absence of any significant amount of bile acids suggests a possible irreversible effect of the bile acid on the expression of G proteins and consequently on the cAMP-mediated signal-transduction pathway. Changes in the expression of the G proteins have been associated with cellular events including development and differentiation (24, 41), as well as pathological conditions (26, 39) and aging (28). In addition, as suggested for the glucagon and beta -adrenergic receptors (42), specific receptors are preferentially coupled to certain G protein subtypes. Therefore, irreversible alterations of specific G protein subtypes may lead to permanent changes in the respective response of selected hormones.

The relationship between the stimulatory and inhibitory role of glucagon and bile acids, respectively, on cAMP formation, as far as bile acid secretion is concerned, is still unclear. However, although still speculative, different hypotheses can be proposed to explain these mechanisms. Because hormones such as glucagon stimulate hepatocellular bile acid uptake and consequently bile secretion through an increase in cAMP synthesis (12, 25), bile acids may exert their effects on cAMP synthesis through a feedback mechanism. The inhibitory effect of bile acids on the hepatocellular cAMP production could allow for a fine regulation of bile acid uptake and secretion and thus prevent any hepatic accumulation of potentially toxic bile acids. Therefore, the reduction of the hormone-induced cAMP production observed during cholestasis could be part of an initial hepatocellular defense mechanism against any cellular accumulation of cytotoxic bile acids.

In conclusion, the findings of the present study link the attenuated regulation of adenylyl cyclase and cAMP synthesis by hormones and bile acids during cholestasis induced by BDL to decreased expression of G proteins, including the stimulatory G protein Gsalpha S as well as the inhibitory G proteins Gialpha 2 and Gialpha 3. However, these results do not preclude a possible additional alteration of the adenylyl cyclase. Furthermore, the sodium-dependent, but not the sodium-independent, hepatocellular bile acid transport mechanism is drastically reduced or abolished 2 days after BDL. Hardison et al. (20) and Coche et al. (9) have proposed that bile acid uptake may be the rate-limiting step in bile acid secretion. Therefore, the feedback mechanism between cAMP synthesis and bile acid uptake may play a central role in the ultimate regulation of bile secretion. Consequently, the attenuation of both cAMP synthesis and the hepatocellular uptake of bile acids could have important implications in the initial prevention of bile acid accumulation in the hepatocyte during cholestasis.

    ACKNOWLEDGEMENTS

The authors thank Deepak Kashyup, Ajit Verghese, Nikhil K. Garg, and Zaheer Arastu for skillful technical assistance, Dr. Susan Ceryak for helpful discussions during the preparation of the manuscript, Dr. Ajit Kumar for providing us with HeLa cells, and Dr. Thomas P. Sakmar (Rockefeller University, New York, NY) for kindly providing us with the ST-18 antibody for the glucagon receptor, as well as the COS cell plasma membrane fraction, which expresses the glucagon receptor.

    FOOTNOTES

The results of this study were presented in part at the annual meeting of the American Gastroenterological Association in Washington, DC, May 1997.

This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant (NIDDK) DK-46954, as well as by a grant from the American Heart Association (Nations Capitol Affiliate) to B. Bouscarel. T. W. Gettys is supported by NIDDK Grant DK-42486 and by a grant from the American Diabetes Association.

Address for reprint requests: B. Bouscarel, Div. of Gastroenterology and Nutrition, Dept. of Medicine, George Washington Univ. Medical Center, 2300 I St. NW, 523 Ross Hall, Washington, DC 20037.

Received 16 June 1997; accepted in final form 2 February 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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