1 Division of Gastroenterology
and Nutrition, 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
bile acid; ursodeoxycholic acid; adenosine 3',5'-cyclic
monophosphate; isolated hamster hepatocytes; cholestasis
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
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.
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
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 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-Gs 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).
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-subunit
(Gs
S).
The expression of either the large subunit
(Gs
L)
or the common
-subunit remained unchanged. The expression of
Gi
2
and
Gi
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
Gs
S
and Gi
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.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
- (40-52 kDa),
- (35-36 kDa), and
-subunits (10 kDa)
(18). The two Gs
isoforms with
apparent molecular masses of 42-45 kDa
(Gs
S)
and 47-52 kDa
(Gs
L)
are produced by alternative splicing of
Gs
mRNA. These
Gs
isoforms have been
identified in numerous tissues, including the liver (16, 42). The
Gi
proteins have been
classified into three subtypes:
Gi
1,
Gi
2, and
Gi
3
(see Ref. 17 for review). However, whereas the
Gi
1 subtype is expressed in the brain and adipose tissue, only the Gi
2
and
Gi
3
isoforms are detectable in the liver (16).
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C.
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
Gs
S
and
Gs
L,
residues 345-354 of
Gi
2,
residues 345-354 of
Gi
3,
or the peptide sequence residues 127-139 of common
-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.
,
-Gi
2,
and
-Gi
3
or common
-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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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.
|
|
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.
|
Characterization of the expression of
Gs and G
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
Gs
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.
|
|
Characterization of Gi
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,
Gi
1-2
(40 kDa) and
Gi
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 Gi
2
was significantly reduced by 25%, whereas
Gi
3
was reduced by 46% in BDL hamster livers.
|
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
cells1 · 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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DISCUSSION |
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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
GsL
was either not changed or slightly increased, whereas that of
Gs
S
was decreased by 40-50% after BDL.
Recently, Yagami (42) reported that in liver membranes the
-adrenergic receptor was only coupled to
Gs
L,
whereas the glucagon receptor was coupled to both
Gs
L
and
Gs
S.
Therefore, in agreement with these results, the 40-50% decrease
in
Gs
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
-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
GsS
and
Gs
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 Gs
S
has been reduced but not abolished after BDL (Fig. 4,
A-C), fewer
Gs
S
can be activated by glucagon, and therefore, UDCA are still able to
uncouple certain of these receptors and
Gs
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
Gi2
and
Gi
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
(Gi
2
or
Gi
3).
However, the decrease of both
Gi
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
Gi
2
rather than
Gi
3
was responsible for the inhibition of the adenylyl cyclase. Gi
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
Gi
2
protein during BDL could represent that
Gi
pool that possesses the
major inhibitory action; 2) not only
Gi
2
but also
Gi
3
proteins are involved in the inhibition of adenylyl cyclase activity
induced by ANG II; 3) whereas only 25% of the
Gi
2
pool has disappeared, a large portion of the remaining Gi
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
Gs expression in hepatic
membranes isolated from cholestatic rats. Furthermore, although both
studies report the decreased expression of
Gi
3
in hepatic membranes after BDL, in contrast to the present study, an
increase of
Gi
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
Gs
S
and
Gs
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
-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
GsS
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 -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 GsS
as well as the inhibitory G proteins
Gi
2
and
Gi
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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