cAMP increases liver
Na+-taurocholate cotransport
by translocating transporter to plasma membranes
Sunil
Mukhopadhayay1,
M.
Ananthanarayanan2,
Bruno
Stieger3,
Peter J.
Meier3,
Frederick J.
Suchy2, and
M. Sawkat
Anwer1
1 Departments of Medicine and
Pharmacology, Tufts University School of Veterinary Medicine, North
Grafton, Massachusetts 01536;
2 Department of Pediatrics, Mount
Sinai Medical Center, New York, New York 10029; and
3 Division of Clinical
Pharmacology, Department of Medicine, University Hospital, CH-8091
Zurich, Switzerland
 |
ABSTRACT |
Adenosine 3',5'-cyclic
monophosphate (cAMP), acting via protein kinase A, increases transport
maximum of Na+-taurocholate
cotransport within 15 min in hepatocytes (S. Grüne, L. R. Engelking, and M. S. Anwer. J. Biol.
Chem. 268: 17734-17741, 1993); the mechanism of
this short-term stimulation was investigated. Cycloheximide inhibited
neither basal nor cAMP-induced increases in taurocholate uptake in rat
hepatocytes, indicating that cAMP does not stimulate transporter
synthesis. Studies in plasma membrane vesicles showed that taurocholate
uptake was not stimulated by the catalytic subunit of protein kinase A
but was higher when hepatocytes were pretreated with cAMP. Immunoblot
studies with anti-fusion protein antibodies to the cloned
Na+-taurocholate cotransport
polypeptide (Ntcp) showed that pretreatment of hepatocytes with cAMP
increased Ntcp content in plasma membranes but not in homogenates. Ntcp
was detected in microsomes, endosomes, and Golgi fractions, and cAMP
pretreatment resulted in a decrease only in endosomal Ntcp content. It
is proposed that cAMP increases transport maximum of
Na+-taurocholate cotransport, at
least in part, by translocating Ntcp from endosomes to plasma
membranes.
protein kinase A; sodium ion-taurocholate cotransport polypeptide; cycloheximide; rat hepatocytes; plasma membrane vesicles
 |
INTRODUCTION |
CONJUGATED BILE ACIDS such as taurocholate
(TC) are efficiently transported by the liver, and the uptake of TC by
hepatocytes is mediated predominantly via a
Na+-coupled cotransport mechanism
(2, 23). A cDNA encoding for Na+-TC cotransport polypeptide
(Ntcp) has recently been cloned using expression cloning in
Xenopus
laevis oocytes (7, 16), and anti-fusion protein antibodies to the cloned transporter recognize a
50-kDa protein in the plasma membrane of hepatocytes (1, 29). In
addition, the microsomal epoxy hydrolase (a 49-kDa protein different
from Ntcp) has also been proposed to mediate hepatocyte Na+-TC cotransport (30).
In a recent study (15), we demonstrated that adenosine
3',5'-cyclic monophosphate (cAMP), acting via protein
kinase A (PKA), stimulates Na+-TC
cotransport by increasing the maximal transport rate; the effect of
cAMP is potentiated by
Ca2+/calmodulin-dependent
processes and is downregulated by protein kinase C. These results would
indicate that the stimulation of Na+-TC cotransport by cAMP may
involve phosphorylation and/or translocation of the transporter
as suggested for the glucose transporter (18, 28) and the
Na+/H+
exchanger (12). cAMP may also induce transporter synthesis and/or alter driving forces. Because cAMP hyperpolarizes
hepatocytes (9) and Na+-TC
cotransport is electrogenic (20, 32), cAMP-induced hyperpolarization may increase the driving force for the cotransport. With the overall goal of defining the mechanism by which cAMP increases transport maximum, the present study was designed to determine whether the effect
of cAMP is caused by increased synthesis of the transporter, changes in
driving forces, and/or translocation of Ntcp. Results suggest
that cAMP increases transport maximum, in part, by translocating Ntcp
to the plasma membrane.
 |
MATERIALS AND METHODS |
Materials.
TC (Na salt) was purchased from Calbiochem (San Diego, CA).
8-(4-Chlorophenylthio)-adenosine 3',5'-cyclic monophosphate
(CPT-cAMP), N6,2'-O-dibutyryl
adenosine 3',5'-cyclic monophosphate (DBcAMP), aprotinin,
leupeptin, cycloheximide, catalytic subunit of PKA, and collagenase
were obtained from Sigma Chemical (St. Louis, MO).
[24-14C]taurocholic
acid (56 mCi/mmol),
[3H]taurocholic acid
(2.1 Ci/mmol) and
[methoxy-3H]inulin (80 Ci/mmol) were purchased from NEN (Boston, MA). Anti-fusion protein
antibodies to the cloned Ntcp were
prepared as previously described (1, 29). Polyclonal antibody against
and
1-subunit of
Na+-K+-adenosinetriphosphatase
(ATPase) were obtained from Upstate Biotechnology (Lake Placid, NY).
Male Wistar rats (200-300 g) obtained from Charles River
Laboratories served as liver donors.
Hepatocyte preparation.
Hepatocytes were isolated from rat livers using a previously described
collagenase perfusion method (3). Freshly prepared hepatocytes
suspended (100 mg wet wt/ml) in a
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) assay buffer (pH 7.4) containing (in mM) 20 HEPES, 140 NaCl, 5 KCl, 1.0 MgSO4, 1.0 CaCl2, 0.8 KH2PO4,
and 5 glucose were incubated for 30 min at 37°C under air before
initiating studies. Three different types of experiments were conducted
with hepatocytes. 1) To determine
whether cAMP stimulates transporter synthesis, hepatocytes were treated
with cycloheximide followed by determination of TC uptake in the
presence and absence of 100 µM DBcAMP.
2) To determine whether the effect
of cAMP is caused by changes in driving forces, plasma membranes were
isolated from hepatocytes pretreated with 100 µM DBcAMP followed by
determination of TC uptake into membrane vesicles. In addition, to
determine whether PKA directly activates TC uptake, plasma membranes
isolated from untreated hepatocytes were vesiculated in the presence of catalytic subunit of PKA followed by determination of vesicular uptake
of TC. 3) To determine whether cAMP
affects Ntcp content, hepatocytes were treated with 10 µM CPT-cAMP
followed by subcellular fractionation and immunoblot analysis. All
studies were repeated in at least three different cell preparations.
Subcellular fractionation.
Plasma membranes, microsomes, endosomes, and Golgi fractions were
isolated from hepatocytes pretreated with cAMP or buffer. All isolation
steps were carried out at 40°C, and cAMP pretreatment did not
significantly affect marker enzyme enrichment.
Plasma membranes were isolated using a Percoll gradient centrifugation
method (10) as described for hepatocytes (17). Briefly, hepatocytes
were homogenized in a buffer (pH 7.4) containing (in mM) 5 HEPES, 0.5 ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 250 sucrose, 1 phenylmethylsulfonyl fluoride (PMSF), and 2 KF and
10 µg/ml leupeptin and aprotinin followed by centrifugation (20 min
at 4,400 g). The resuspended pellet
was subjected to Percoll gradient centrifugation (10). The membrane
fraction collected at the buffer-10% Percoll interface was recovered,
washed twice in the homogenization buffer, and stored at
70°C. It may be noted that plasma membrane fractions
obtained by this method (10) contain both sinusoidal and canalicular
membranes. Plasma membrane fractions so derived were enriched in
5'-nucleotidase and
Na+-K+-ATPase,
and the enrichment was not significantly affected by cAMP pretreatment
(Table 1).
Microsomes were isolated using a differential centrifugation method as
described previously (22). These microsomes were enriched in
glucose-6-phosphatase, NADH dehydrogenase, and 5'-nucleotidase by
3-, 4-, and 0.35-fold, respectively.
Endosome and endosome-depleted Golgi fractions were isolated using a
sucrose-gradient centrifugation method (11). Briefly, hepatocytes were
washed in HEPES assay buffer followed by resuspension in an isolation
buffer (pH 7.4) containing (in mM) 10 tris(hydroxymethyl)aminomethane (Tris) · HCl, 2 CaCl2, 5 MgCl2, 1 PMSF, and 2 KF with 3 µg/ml leupeptin and 2 µg/ml aprotinin, homogenization (glass
homogenizer with 10 strokes), and centrifugation at 1,000 g for 10 min. The resulting pellet was
washed twice with the isolation buffer, and the pooled supernatant
fraction (S1) was centrifuged at 33,000 g for 8 min. The resulting supernatant
fraction (S2) was used to isolate endosome fractions, and the loose
region of the pellet was used to isolate Golgi fractions. The loose
region of the pellet was collected by swirling in 72% sucrose. After
dispersion of the loose region into a loose-fitting Dounce homogenizer,
the sucrose concentration was adjusted to 39% and overlayered with an
equal volume of 29.5, 20.5, and 8% sucrose in ultracentrifuge tubes
followed by centrifugation at 97,000 g
for 3 h. The Golgi fraction at the interface of 29.5 and 39% sucrose
was collected, washed, and stored at
70°C. The S2 fraction
was centrifuged at 160,000 g for 45 min, and the pellet containing "crude endosomes" was resuspended
in the isolation buffer, washed twice, and stored at
70°C.
Further subfractionation of crude endosomes was carried out by loading
S2 fraction onto sucrose gradient consisting of 1 ml 70% sucrose, 5 ml
of 43% sucrose, and a continuous gradient by mixing 7.5 ml 40% and
7.5 ml 15% sucrose and centrifuging at 97,000 g for 4 h. Three fractions designated
as fractions 1,
2, and
3 (corresponding to
fractions
D, E,
and P in Ref. 11) were obtained;
fractions 1 and
2 represent low-density endosomes, and fraction 3 is of unknown origin
(11). Fraction 3, but not
fractions 1 and
2, was enriched in
5'-nucleotidase (2-fold) and
Na+-K+-ATPase
(3-fold), indicating presence of plasma membranes (Table 1).
Pretreatment with cAMP did not result in a significant difference in
the enrichment of these marker enzymes.
TC uptake in hepatocytes and plasma membrane vesicles.
The initial uptake rate of TC in hepatocytes was determined as
previously described (2). Briefly, at various times after incubation of
hepatocytes with cycloheximide and/or DBcAMP, an aliquot of
cell suspension (5-8 mg protein/ml) was withdrawn and used to
determine the initial uptake rate of TC (20 µM). Transport was
initiated by adding cells to the incubation medium containing [14C]TC and
[3H]inulin, with
uptake determined at different time points. Initial uptake rates were
calculated from the slope of the linear portion of time-dependent
uptake curves and were expressed in nanomoles per minute per milligram
of protein.
A rapid filtration technique was used to determine TC uptake in plasma
membrane vesicles (6). Briefly, frozen plasma membrane suspensions were
rapidly thawed by immersion in a 37°C water bath, diluted to the
desired protein concentration (2-4 mg/ml), and vesiculated by
passing membranes 20 times through a 27-gauge needle. To study the
effect of PKA on TC uptake, membranes were suspended in a buffer
containing the catalytic subunit of PKA (50 U/ml) and ATP (1.0 mM)
before vesiculation. An aliquot (20 µl) of membrane vesicles was
incubated for 15 min at 37°C, and the uptake was initiated by
adding 80 µl of incubation buffer (in mM: 100 sucrose, 100 NaCl, 0.2 CaCl2, 5 MgSO4, and 20 HEPES-Tris, pH 7.5)
containing [3H]TC (2.5 µCi/ml or 1.19 µM). The uptake was terminated by the addition of
3.5 ml of ice-cold stop solution (in mM: 100 sucrose, 100 KCl, 0.2 CaCl2, 5 MgSO4, and 20 HEPES-Tris, pH 7.5).
Membrane vesicles were separated from the incubation medium by
immediate filtration through a 0.45-µm Millipore filter (HAWP) that
was presoaked in a buffer containing 1 mM TC. The filter was washed twice with 3.5 ml of stop solution, dissolved in 10 ml of scintillation fluid, and counted for radioactivity. Nonspecific binding to membranes was determined using the same method except that the procedure was
conducted at 0-4°C. The uptake was calculated from
membrane-associated radioactivity after subtracting nonspecific binding
and was expressed in picomoles per milligram of protein.
Immunoblot analysis.
Proteins from different subcellular fractions were subjected to 12%
sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis by the
method of Laemmli (19). Proteins were transferred electophoretically from SDS gels to nitrocellulose membranes (Transblot, transfer membrane
0.45 µm; Bio-Rad) and probed with the appropriate antibody (anti-Ntcp
at 1:2,000 dilution or
anti-
1-subunit of
Na+-K+-ATPase
at 1:1,000 dilution). Peroxidase-conjugated anti-immunoglobulin G was
used as the secondary antibody. The immunoblots were developed with the Amersham enhanced chemiluminescence kit according to the
manufacturer's instructions, and a laser densitometer (UltroScan XL,
Pharmacia) was used to obtain relative quantitation of the signals.
Other methods.
The Lowry method was used to determine cell protein (21). Marker
enzymes 5'nucleotidase (4),
Na+-K+-ATPase
(27), NADH dehydrogenase (31), and glucose-6-phosphatase (4) were
assayed using established methods. All values are expressed as means ± SE. Student's t-test (or paired
t-test) was used to statistically
analyze data, with P < 0.05 considered significant.
 |
RESULTS |
Effect of cAMP on transporter synthesis.
The effect of cycloheximide on TC uptake was studied in isolated
hepatocytes to determine whether the stimulatory effect of cAMP
involves new protein synthesis. Neither basal nor cAMP-stimulated TC
uptake was affected by cycloheximide (50 µg/ml) for up to 120 min
(Fig. 1). Thus it is unlikely that cAMP
stimulates Na+-TC cotransport by
increasing transporter synthesis.

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Fig. 1.
Effect of cycloheximide on basal and adenosine 3',5'-cyclic
monophosphate (cAMP)-induced taurocholate (TC) uptake. Hepatocytes were
treated with cycloheximide (50 µg/ml) and/or 100 µM
N6,2'-O-dibutyryl
adenosine 3',5'-cyclic monophosphate (DBcAMP), and TC (20 µM) uptake was determined at indicated times. Data represent means ± SE; n = 5 different cell
preparations. Pre, before treatment.
|
|
Effect of PKA and cAMP pretreatment on TC uptake in plasma membrane
vesicles.
PKA may directly affect the transporter and thereby stimulate
Na+-TC cotransport. To test this,
the effect of catalytic subunit of PKA on TC uptake in membrane
vesicles obtained from untreated hepatocytes was determined. A similar
approach was employed by Bae and Verkman (5) to study the regulation of
Cl
conductance in endocytic
vesicles by cAMP. TC uptake in membrane vesicles containing PKA and ATP
was not significantly different from control values (Fig.
2). Thus PKA may not directly alter the
transporter activity. To ascertain that PKA was active under the
experimental conditions used, plasma membranes were similarly treated
with PKA and [32P]ATP,
and membrane proteins were subjected to SDS-gel electrophoresis followed by autoradiography. Presence of multiple phosphorylated proteins on the gel (data not shown) indicated that PKA was active.

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Fig. 2.
TC uptake in plasma membrane vesicles.
Top: plasma membranes isolated from
untreated hepatocytes were vesiculated in presence of catalytic subunit
of protein kinase A (PKA; 50 U/ml) and 1.0 mM ATP (PKA + ATP) or buffer
(control) before uptake studies. Data represent means ± SE;
n = 3 membrane preparations.
Bottom: plasma membranes were isolated
from hepatocytes pretreated with 100 µM DBcAMP (cAMP) or buffer
(control). Data represent means ± SE;
n = 4 membrane preparations.
* Significantly different from respective control values by
paired t-test (average values of
triplicate determinations for each membrane preparation were paired).
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cAMP may stimulate Na+-TC
cotransport by increasing driving forces and/or by inducing
stable changes in the transporter at the plasma membrane level. In the
former case, plasma membranes isolated from cAMP-treated hepatocytes
should not show increased TC uptake. This is because the driving force
(Na+ gradient) for TC uptake in
plasma membrane vesicles is controlled by the incubation medium.
However, plasma membrane vesicles should retain the stimulatory effect
of cAMP, if cAMP stably changes the transporter in the plasma membrane.
Thus TC uptake was determined in plasma membrane vesicles isolated from
hepatocytes pretreated with cAMP (Fig. 2). The initial rate of TC
uptake was 40% higher (8.6 ± 1.3 vs. 6.1 ± 0.95 pmol · 10 s
1 · mg
protein
1; means ± SE;
n = 4) in vesicles obtained from
cAMP-treated hepatocytes. Thus the stimulation of
Na+-TC cotransport by cAMP may
involve stable changes in the transporter.
Effect of cAMP on plasma membrane Ntcp content.
cAMP may increase the maximum uptake rate of TC by increasing plasma
membrane content of Ntcp. Because Ntcp has been shown to mediate
Na+-TC cotransport (27),
anti-fusion protein antibodies to the cloned
Ntcp were used to determine whether
cAMP increases plasma membrane content of Ntcp. Two different
antibodies from two different laboratories (1, 29) were used. Because
results obtained with these antibodies were similar, they are not
reported separately. For these studies, hepatocytes were treated with
cAMP followed by isolation of plasma membranes. Immunoblot analysis
showed that cAMP treatment resulted in a 50% increase in the amount of
Ntcp in the plasma membrane (Fig. 3). cAMP
did not alter the amount of Ntcp in the whole homogenate (Fig. 3),
indicating that the increase in plasma membrane content is not caused
by an overall increase in Ntcp. We have also determined the effect of
cAMP on plasma membrane content of organic anion transporting protein (Oatp) and the
1-subunit of
Na+-K+-ATPase.
Treatment of hepatocytes with cAMP did not result in an increase in
plasma membrane content of either Oatp or the
1-subunit of
Na+-K+-ATPase
(Fig. 4). Immunoblot analysis of Oatp using
our plasma membrane preparations was conducted in Dr. A. Wolkoff's
laboratory. Thus it is unlikely that the increase in Ntcp is caused by
a general effect of cAMP on distribution of proteins in plasma
membranes. This observed increase in plasma membrane content of Ntcp
raises the possibility that cAMP-induced stimulation of
Na+-TC cotransport may involve
translocation of the transporter from intracellular stores to the
plasma membrane.

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Fig. 3.
Effect of cAMP on Na+-TC
cotransport polypeptide (Ntcp) content of plasma membranes and
homogenate prepared from hepatocytes pretreated with 10 µM
8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate
(CPT-cAMP; cAMP) or buffer (con) for 15 min.
Top: immunoblot analysis of Ntcp in
plasma membranes and homogenate using 20 (left) and 60 (right) µg protein.
Bottom: densitometric analysis. Data
represent means ± SE; n = 4 different preparations. * Significantly different from respective
control values.
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Fig. 4.
Effect of cAMP on content of organic anion transporting protein (Oatp)
and 1-subunit of
Na+-K+-ATPase
in plasma membranes isolated from hepatocytes pretreated with 10 µM
CPT-cAMP (cAMP) or buffer (Con) for 15 min. Band intensities relative
to respective controls were 1.09 ± 0.18 and 0.95 ± 0.11 (means ± SD) for Oatp (n = 3) and
Na+-K+-ATPase
(n = 5), respectively.
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Effect of cAMP on subcellular distribution of Ntcp.
To determine intracellular source(s), hepatocytes were treated with
cAMP followed by isolation of plasma membrane, microsome, endosome, and
Golgi fractions. Immunoblot analysis showed that Ntcp antibody
recognized more than one protein band in the general area of 50 kDa
(Fig. 5), possibly representing different glycosylated forms (23).
Densitometric analysis showed that cAMP-induced increase in plasma
membrane content of Ntcp was not associated with a significant decrease
in microsomes (data not shown), crude endosomes, and Golgi fractions
(Fig. 5). To determine whether a subpopulation of endosomes may be the intracellular source, the crude
endosomal fraction was further subfractionated on sucrose gradient and
the resulting three fractions (fractions
1-3) were subjected to immunoblot analysis (Fig.
6). Results showed that cAMP
decreased Ntcp content in fraction
2 and increased Ntcp content in
fraction
3 without significantly affecting Ntcp
content in fraction
1. Thus cAMP may stimulate
translocation of Ntcp from endosomes to plasma membranes.

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Fig. 5.
Effect of cAMP on Ntcp content of plasma membranes, crude endosomes,
and Golgi fraction isolated from hepatocytes pretreated with 10 µM
CPT-cAMP (cAMP) or buffer (Con) for 15 min.
Top: immunoblot analysis of Ntcp in
plasma membranes, crude endosomes, and Golgi fraction using 30, 20, and
30 µg protein, respectively. Bottom:
densitometric analysis. Data represent means ± SE;
n = 3 different
preparations. * Significantly different from respective control
values.
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Fig. 6.
Effect of cAMP on Ntcp content of various endosome fractions. Crude
endosomes isolated from hepatocytes pretreated with 10 µM CPT-cAMP
(cAMP) or buffer (Con) for 15 min were subfractionated on sucrose
gradient. Top: immunoblot analysis of
Ntcp in endosome fractions 1,
2, and
3 using 13, 30, and 38 µg protein,
respectively. Bottom: densitometric
analysis. Data represent means ± SE;
n = 4 different preparations.
* Significantly different from respective control values.
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 |
DISCUSSION |
cAMP is known to stimulate Na+-TC
cotransport in hepatocytes by increasing transport maximum within 15 min. The present study was designed to determine whether this rapid
effect of cAMP is caused by increased synthesis of the transporter(s),
changes in driving forces, and/or translocation of Ntcp.
Results showed that the stimulatory effect of cAMP was not affected by
cycloheximide and was still present in plasma membranes isolated from
hepatocytes pretreated with cAMP. In addition, cAMP treatment of
hepatocytes resulted in an increase in plasma membrane and a decrease
in low-density endosomal Ntcp content. These results are discussed in
relation to our conclusion that cAMP increases the maximum transport
rate of Na+-TC cotransport, in
part, by translocating Ntcp from endosomes to the plasma membrane.
The stimulatory effect of cAMP is unlikely to be caused by increased
synthesis of the transporter protein, because cycloheximide (50 µg/ml) failed to inhibit the effect of cAMP (Fig. 1). Moreover, the
stimulatory effect of cAMP is maximal within 30 min (Fig. 1) and new
protein synthesis usually requires a longer time interval. The same
concentration of cycloheximide has been shown to inhibit long-term
(>30 min) but not short-term stimulation of
L-alanine uptake by glucagon in
hepatocytes (8), an effect mediated via cAMP. The long-term stimulation
of L-alanine transport by cAMP is believed to be caused by stimulation of transporter synthesis. Because cAMP-stimulated TC uptake was not affected by cycloheximide for
120 min, it is likely that cAMP regulates
Na+-TC cotransport differently
than L-alanine transport in
hepatocytes. The stimulatory effect of cAMP was retained in plasma
membrane vesicles isolated from cAMP-pretreated hepatocytes (Fig. 2).
This result would indicate that the effect of cAMP may be independent of cAMP-induced changes in driving forces. However, this result does
not rule out the possibility that the effect of cAMP in hepatocytes may, in part, be mediated via changes in the driving force. Studies in
hepatocytes indicate that Na+-TC
cotransport is electrogenic (20, 32), and cAMP has been shown to
hyperpolarize hepatocytes (9). Thus cAMP is likely to stimulate
Na+-TC cotransport by increasing
the electrical gradient, as suggested by our previous preliminary
studies (14). The retention of the stimulatory effect in plasma
membrane vesicles would then imply that cAMP stimulates
Na+-TC cotransport by inducing
stable changes at the plasma membrane level in addition to changes in
driving forces. On the basis of similar studies in plasma membranes
isolated from pretreated hepatocytes, it was concluded that cAMP also
induces stable changes in the L-alanine transporter at the
plasma membrane level (26).
One mechanism by which cAMP can induce stable changes that will result
in increased maximum transport activity is increasing the number of
transporter in the plasma membrane. This possibility is
supported by our result that cAMP increases the Ntcp content in plasma
membranes (Fig. 3). Because Ntcp has been shown to mediate Na+-TC cotransport (23), it is
most likely that cAMP-induced change in Ntcp is involved in the
regulation of this cotransporter in hepatocytes. cAMP did not affect
the Ntcp content of the whole homogenate, indicating that the increase
in plasma membrane content is not caused by an increase in Ntcp
synthesis. This result is consistent with our finding that
cycloheximide did not affect the stimulatory effect of cAMP. This
result also suggests that the increase in plasma membrane content is
likely to be caused by translocation of the transporter from
intracellular stores.
The present study also showed that the increase in plasma membrane Ntcp
by cAMP was associated with a decrease in endosomal fraction 2 (Fig. 6). This result may
indicate that cAMP stimulates translocation of Ntcp from endosomes to
the plasma membrane. However, cAMP also increased Ntcp content of
fraction 3 (Fig. 6), raising the
possibility that these changes may result from redistribution of Ntcp
in different endosomal compartments by cAMP. Although this possibility
cannot be ruled out, it seems unlikely for the following reasons.
Fractions 1-3 were obtained when
the crude endosomal preparation was further subfractionated (see
MATERIALS AND METHODS). Although
fractions 1 and
2 represent early endosomes, the
origin of fraction 3 is unknown and
may not represent endosomes (fraction
P in Ref. 11). Determination of marker enzyme activity showed that fraction 3 is enriched in
5'-nucleotidase and
Na+-K+-
ATPase (Table 1), indicating the presence of canalicular as well as
sinusoidal plasma membranes in this fraction. On the other hand, the
activities of these enzymes were not higher in
fractions 1 and
2 compared with homogenate (Table 1).
Thus it is possible that the observed increase in the Ntcp content of
fraction 3 is caused by the presence
of basolateral membranes. On the basis of these results, we propose
that cAMP stimulates Na+-TC
cotransport in part by translocating Ntcp, most likely from endosomes,
to plasma membranes. Regulation of transport activity by translocation
has been proposed for glucose transporter. For example, the ability of
insulin to activate glucose transport in adipocytes and myocytes is
mainly caused by translocation of GLUT-4 to plasma membranes (28), and
cAMP has also been shown to acutely stimulate translocation of glucose
transporter (GLUT-4) from low-density microsomal membranes to the
plasma membranes in rat adipocytes (18).
The activity of a transporter can also be regulated by phosphorylation
(12). Whether cAMP also regulates
Na+-TC cotransport by altering
phosphorylation status is not known. Our preliminary study indicates
that Ntcp is a phosphoprotein (25). Because PKA failed to stimulate TC
uptake in plasma membrane vesicles (Fig. 2), it is unlikely that the
stimulation of Na+-TC cotransport
by cAMP is caused by PKA-mediated direct phosphorylation of the
transporter. This, however, does not rule out regulation by
phosphorylation/dephosphorylation, because the phosphorylation status
of the transporter may be affected by kinases and phosphatases that
are, in turn, regulated by PKA. The potential role of phosphorylation is currently under investigation. In the present study, antibodies against Ntcp were used to determine the effect of cAMP on
Na+-TC cotransporter protein.
However, another protein (microsomal epoxy hydrolase) unrelated to Ntcp
has been proposed to mediate plasma membrane
Na+-TC cotransport (30). Whether
cAMP also affects this protein remains to be determined. It may,
however, be mentioned that Ntcp appears to represent the major
Na+-TC cotransport system in the
rat liver (23), and Ntcp has been shown to be downregulated in
cholestasis associated with decreased Na+-TC cotransport (13, 24).
In summary, the present study showed that the stimulation of
Na+-TC cotransport in hepatocytes
by cAMP is unlikely to be caused by synthesis or PKA-mediated direct
phosphorylation of the transporter. It is proposed that cAMP increases
the transport maximum by translocating Ntcp from intracellular stores
to the plasma membrane.
 |
ACKNOWLEDGEMENTS |
The authors gratefully acknowledge the excellent technical
assistance of Holly Jameson. The authors also thank Dr. I. M. Arias and
L. R. Engelking for helpful discussion and Dr. A. Wolkoff for
immunoblot analysis of Oatp on our plasma membrane preparations.
 |
FOOTNOTES |
This study was supported in part by National Institutes of Health
Grants DK-33436 (M. S. Anwer) and HD-20632 (F. J. Suchy) and by Swiss
National Foundation Grants 32-2987890 (P. J. Meier) and
31-33520-92 (B. Stieger).
Address for reprint requests: M. S. Anwer, Depts. of Medicine and
Pharmacology, Tufts Univ. School of Veterinary Medicine, 200 Westboro
Rd., North Grafton, MA 01536.
Received 9 January 1997; accepted in final form 23 June 1997.
 |
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