Mechanisms by which cAMP increases bile acid secretion in rat liver and canalicular membrane vesicles
Suniti Misra,
Lyuba Varticovski, and
Irwin M. Arias
Department of Physiology, Tufts University School of Medicine, Boston,
Massachusetts 02111
Submitted 29 January 2003
; accepted in final form 11 April 2003
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ABSTRACT
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Bile acid secretion induced by cAMP and taurocholate is associated with
recruitment of several ATP binding cassette (ABC) transporters to the
canalicular membrane. Taurocholate-mediated bile acid secretion and
recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K)
dependent and require an intact microtubular apparatus. We examined mechanisms
involved in cAMP-mediated bile acid secretion. Bile acid secretion induced by
perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and
wortmannin, a PI3K inhibitor. Canalicular membrane vesicles isolated from
cAMP-treated rats manifested increased ATP-dependent transport of taurocholate
and PI3K activity that were reduced by prior in vivo administration of
colchicine or wortmannin. Addition of a PI3K lipid product, phosphoinositide
3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate,
restored ATP-dependent taurocholate in these vesicles. Addition of a
decapeptide that activates PI3K to canalicular membrane vesicles increased
ATP-dependent transport above baseline activity. In contrast to effects
induced by taurocholate, cAMP-stimulated intracellular trafficking of the
canalicular ABC transporters was unaffected by wortmannin, and recruitment of
multidrug resistance protein 2, but not bile salt excretory protein (bsep),
was partially decreased by colchicine. These studies indicate that trafficking
of bsep and other canalicular ABC transporters to the canalicular membrane in
response to cAMP is independent of PI3K activity. In addition, PI3K lipid
products are required for activation of bsep in the canalicular membrane.
These observations prompt revision of current concepts regarding the role of
cAMP and PI3K in intracellular trafficking, regulation of canalicular bsep,
and bile acid secretion.
ATP binding cassette transporters; bile acid secretion; bile salt export protein; multidrug resistance protein 2; phosphatidylinositol 3-kinase
THE ENTEROHEPATIC CIRCULATION of bile acids efficiently provides
for their availability in response to requirements for emulsification of
dietary fats and results in postprandial elevations of plasma bile acids
(4). The hepatic response is
rapid and includes enhanced bile acid uptake, transcellular transport, and
subsequent secretion in to the bile. These processes and unresolved issues
regarding bile acid secretion and intracellular transport have been reviewed
elsewhere (4,
43,
51). Intracellular trafficking
of canalicular ATP binding cassette transmembrane proteins (ABC transporters)
requires complex motor-driven vesicular movement along microtubules,
phosphatidylinositol 3-kinase (PI3K) lipid products, small GTPases, and other
unidentified components (21,
37). Bile salt excretory
protein (bsep), which is also termed abcb11, is an ABC transporter that is
required for ATP-dependent secretion of bile acids across the canalicular
membrane (12). Of importance
to the present study is the observation that, on the basis of kinetic analysis
of transport rates using isolated canalicular membrane vesicles (CMV), bsep
should be saturated at basal bile acid concentrations
(30). Obviously, this could
not account for rapid postprandial increments in bile acid secretion or
choleretic responses that immediately follow intravenous or oral
administration of taurocholate (TC) and choleretic agents, including cAMP
(4,
9,
13,
21). Recent studies
demonstrate that bsep and other canalicular ABC transporters, such as
multidrug resistance (mdr) 1 and 3, and multidrug resistance protein 2 (mrp2),
traffic from Golgi directly to the canalicular membrane
(20,
39). Recruitment of these
transporters to the canalicular membrane in response to TC and dibutyryl cAMP
(dBcAMP), a soluble analog of cAMP, is rapid, is reversible, occurs in the
absence of new protein synthesis, and is additive, which suggests that
different pathways may be involved
(11,
22,
27). These responses do not
involve enhanced transcription or translation, which have been elucidated as
regulatory determinants of sterol synthesis, secretion, absorption, and
transport (25). The effects of
TC on intracellular trafficking and bile secretion are reduced by pretreatment
with colchicine, a microtubular inhibitor, and by wortmannin (WM), an
inhibitor of PI3K (11,
27). In addition, the activity
of several ABC transporters recruited in response to TC to the canalicular
membrane is regulated by PI3K lipid products and restored by incubation of CMV
with 3'-phosphoinositides in the presence of WM
(28). These observations
indicate that TC increases microtubule-dependent trafficking of ATP-dependent
transporters and that bile acid secretion requires activation of PI3K. In
WIF-B9 cells, cAMP and TC stimulated PI3K activity through a
phosphotyrosine-independent mechanism in association with increased bile acid
secretion (19).
PI3K and its 3'-polyphosphoinositide lipid products regulate many
biological responses, including receptor-initiated mitogenesis, vesicular
trafficking, oxidative burst, membrane ruffling, glucose uptake, and
activation of membrane ion channels
(3,
7). Typically, mitogens that
activate protein tyrosine kinases stimulate type I PI3K-
or -
,
whereas those that activate G protein-coupled receptors lead to activation of
PI3K-
(3,
46); however, exceptions have
been noted (29,
35). Less is known about
mechanisms responsible for PI3K activation by cAMP. Although cAMP-stimulated
PI3K and p70 S6 kinase are sufficient to stimulate cell cycle progression
(6,
34), there are additional cell
type-specific mechanisms by which cAMP stimulates PI3K activity,
proliferation, and bile acid secretion
(6,
19,
34,
48).
The present studies were performed to identify mechanisms by which cAMP
increases bile acid secretion and intracellular trafficking of ABC
transporters, particularly in relation to PI3K activity in CMV. Results were
compared with those described after treatment with TC
(11,
27).
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MATERIALS AND METHODS
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Reagents. Tris base, EDTA, CaCl2, sucrose, HEPES, TC,
WM, colchicine, protein A-Sepharose beads,
L-leucine-p-nitroanilide,
L-glutamyl-p-nitroanilide, dBcAMP, and all other reagents
were purchased from Sigma-Aldrich (St. Louis, MO) and were of highest purity.
A synthetic rhodamine-linked membrane-permeable 10-mer peptide based on the
phosphoinositide-binding sequence of gelsolin and designated as PI3K peptide
was a gift of P. Janmey (University of Pennsylvania) and J. Hartwig (Brigham
and Women's Hospital, Boston, MA)
(17,
18). [3H]TC
(25 Ci/mmol) and [
-32P]ATP (6,000 Ci/mmol) were from
Perkin Elmer (Boston, MA). EAG15 (polyclonal anti-mrp2) was a gift from D.
Keppler (University of Heidelberg, Heidelberg, Germany); K12 (polyclonal
anti-bsep) was a gift from B. Stieger (University of Zurich, Zurich,
Switzerland). Polyclonal anti-P110
antibody was from Santa Cruz
Biotechnology (Santa Cruz, CA). Polyclonal anti-p110
and p110
and
monoclonal anti-phosphotyrosine antibodies were from Upstate Biotechnology
(Lake Placid, NY).
In situ perfusion of the rat liver. The procedures using animals
were approved by the Institutional Animal Care and Use Committee. Male
Sprague-Dawley rats weighing 250300 g were purchased from Charles River
Farms (Wilmington, MA). Rats were anesthetized with sodium pentobarbital (50
mg/kg), and nonrecirculating single-pass liver perfusion was performed in situ
according to Hems et al. (14).
Liver viability was sustained by maintaining portal pressure (average 10
cmH2O), O2 supply, temperature (37°C), and buffer pH
(7.357.40) throughout the perfusion. Animals were perfused at 30 ml/min
with 95% O2-5% CO2 oxygenated Krebs-Ringer-bicarbonate
buffer containing 5.5 mM glucose and 10 µM TC for 10 min as described
(11,
16). [3H]TC (2
x 107 cpm/ml) was added to the perfusate 10 min later. The
effect of dBcAMP on TC secretion and bile volume was determined following
addition of 100 µM dBcAMP in the perfusion buffer at indicated times. The
effect of WM on TC- or dBcAMP-induced bile acid secretion was determined
following addition of 100 nM WM to the buffer before or after perfusion with
dBcAMP as indicated. Colchicine was administered intraperitoneally over 1 min
at 2.5 mg/kg in 0.5 ml of PBS at 2.5 h before perfusion. No toxicity to
colchicine was apparent, and respiration and body temperature were maintained.
Bile was collected every 3 min, samples were weighed to determine volume, and
TC secretion was quantified. Immediately following perfusion, the liver was
removed for preparation of CMV.
In vivo treatment of rats. For these experiments, rats received a
single injection of PBS (control), 100 µmol dBcAMP or TC into the tail vein
in a 0.5 ml PBS over 1 min, or a single intraperitoneal injection of
colchicine at 2.5 mg/kg in 1 ml of PBS at 45 min before preparation of CMV or
liver extracts.
Preparation of CMV. After removal from control and experimental
animals, the liver was perfused briefly at room temperature with 0.25 M
sucrose, 10 mM HEPES-Tris with protease inhibitors (in µg/ml: 2 aprotinin,
2 leupeptin, 2 pepstatin, 100 phenylmethylsulfonyl fluoride, and 5
benz-amidine) to remove blood, minced, and homogenized in 5 volumes of buffer.
CMV were isolated from liver homogenates following nitrogen cavitation and
Ca2+ precipitation as described
(16). Vesicle purity was
determined by using leucine aminopeptidase
(38) and
-glutamyl
transpeptidase as markers
(32). With respect to activity
in CMV compared with homogenate, enrichment was 50- to 70-fold with leucine
aminopeptidase and 20- to 30-fold with
-glutamyl transpeptidase. The
yield of CMV was 11.2 mg protein/60 g rat liver. CMV were stored in
aliquots in buffer A (10 mM HEPES-Tris, pH 7.4, 0.25 M sucrose, 0.2
mM CaCl2) at -70°C until use.
Bile acid transport studies. Transport of TC was measured by an
optimized rapid filtration method
(16,
30). The reaction mixture
contained 1.2 mM ATP, an ATP-regeneration system (3 mM creatine phosphate, 100
µg/ml creatine kinase) in buffer B (10 mM HEPES-Tris, pH 7.4, 0.25
M sucrose, 10 mM MgCl2, 0.2 mM CaCl2), and 10 µM TC,
which contained a trace amount of [3H]TC. For each time point,
transport was initiated by adding 20 µl of reaction mixture, which had been
prewarmed for 5 min at 37°C, to CMV (2040 µg protein) suspended
in buffer A at 37°Cina final volume of 50 µl. After 1 min, the
reaction was stopped by addition of 1 ml of ice-cold buffer B.
Vesicles were filtered through glass microfiber filters (Whatman 0.45 µM)
and washed twice with 10 ml of ice-cold buffer B. Radioactivity on
the filters was measured by using a liquid scintillation counter (Beckman LS
1801). Phosphatidylcholine at 0.2 mM final concentration alone or in
combination with 20 µM phosphoinositide 3,4-bisphosphate (PI
3,4-P2) or phosphoinositide 4,5-bisphosphate (PI 4,5-P2)
was sonicated in 10 mM HEPES-Tris, pH 7.4, immediately before use. CMV were
incubated at 37°C for 10 min with the lipids as indicated or for 20 min
with PI3K peptide. Transport assays were performed in triplicate samples.
PI3K activity assay. Assays were performed using 250 ng of CMV
solubilized in 0.5% NP-40. The assay was performed as previously described in
a total volume of 50 µl using 150 µM ATP, 125 mM MOPS, pH 7.0, 25 mM
MgCl2, 5 mM EGTA, and 0.2 mg/ml sonicated lipids: phosphatidyl
serine/PI/PI 4,5-P2 (1:1:1) in sonication buffer (25 mM MOPS, pH
7.0, and 1 mM EGTA) with 15 µCi of [
-32P]ATP per assay
(42). Assays proceeded at
37°C for 20 min and were stopped with 100 µl MEOH:1 N HCl (1:1), and
lipids were extracted twice with 100 µl of chloroform. The organic layer
was combined, dried under nitrogen gas, and analyzed by TLC.
32P-labeled phosphoinositides were resolved in water/acetic
acid/n-propanol (34:1:65) and detected by autoradiography. 32P
incorporation into phosphoinositide 3,4,5-trisphosphate was quantified by
liquid scintillation counting of TLC spots that were scraped and eluted in
scintillation fluid.
CMV extracts and immunoprecipitation. CMV were lysed in buffer
that contained 0.5% NP-40, 50 mM HEPES, pH 7.5, 50 mM sodium fluoride, 5 mM
sodium orthovanadate, 0.5 mM EGTA, 10% glycerol, and protease inhibitors
(leupeptin and aprotinin at 10 µg/ml, pepstatin at 5 µg/ml, and PMSF at
0.5 mM) and centrifuged at 12,000 g for 10 min. The supernatant was
separated, frozen immediately in aliquots by immersion in liquid nitrogen, and
stored at -70°C until use. Immune precipitates were performed using
300400 µg of cell lysate protein from control and experimental
livers in a 300-µl volume incubated with primary antibody for 24 h
and with protein A-Sepharose beads for the last hour of incubation. After
beads were washed once with lysis buffer and three times with PBS, the immune
complexes were sedimented by brief centrifugation and used for PI3K assay as
above.
Immunoblots. Protein (2550 µg protein/lane) was
denatured and resolved on a 10% polyacrylamide gel by using a Bio-Rad minigel
apparatus. Proteins were transferred to nitrocellulose membranes and blocked
for 1 h with PBS containing 7% nonfat milk, 2% BSA, and 0.1% Tween 20.
Membranes were washed and probed with the primary antibodies followed by
secondary antibodies conjugated to horse-radish peroxidase. Immune complexes
were detected by enhanced chemiluminescence reagent according to the
manufacturer's instructions and quantified by densitometry
(27). Membranes were reused
for blotting after neutralization with 15% H2O2 or
stripping at 50°C.
Statistical analysis. Statistical analysis of data was performed
by ANOVA using a Bonferroni test for multiple variants or unpaired
t-test for individual variants. Results with statistical significance
of P < 0.05 at 95% confidence index are indicated by asterisks in
Figs. 2,
3,
4,
5,
6.

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Fig. 2. Effect of WM and colchicine on cAMP-mediated recruitment of bile salt
excretory protein (bsep) and multidrug resistance protein (mrp2) to the
canalicular membrane. The content of mrp2 (open bars) and bsep (gray bars) in
canalicular membrane vesicles (CMV) was quantified by Western blotting. CMV
were prepared from control or rat liver perfused with WM (A) or
pretreated with colchicine (B) in addition to dBcAMP. The
experimental design is described in Fig.
1. One of three representative experiments is shown. Bands
visualized by Western blot were quantified by densitometry. Results were
expressed as %values obtained from buffer-perfused liver (control) and are
shown at bottom (n = 3). Statistical analysis of the data
was performed using ANOVA and Bonferroni test of multiple variants or unpaired
t-test of individual variants. *P < 0.05 at
95% confidence index. **Statistically significant differences that were
calculated by t-test.
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Fig. 3. Effect of WM and colchicine on cAMP-induced phosphoinositide 3-kinase
(PI3K) activity. A: total PI3K activity was measured in solubilized
CMV isolated from liver after perfusion with control buffer, WM, dBcAMP,
dBcAMP followed by WM, and dBcAMP after WM. B: effect of colchicine
on PI3K activity in CMV from control and dBcAMP-treated rats as presented in
Fig. 1C legend. PI3K
activity in different preparations of CMV from control animals (n =
6) varied between 2.0 and 4.0 x l0-3 cpm/mg
protein. Results are expressed as %control ± SD in 3 independent
experiments performed in duplicate. Statistical analysis was performed as in
Fig. 2.
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Fig. 4. Effect of dBcAMP and PI3K lipid products on ATP-dependent TC transport in
CMV. A: ATP-dependent transport in CMV isolated from liver perfused
with buffer (open bar), dBcAMP (gray bar), WM (closed bar), WM added following
dBcAMP (hatched bar), or WM followed by cAMP (blocked bar). TC transport was
also measured in the same preparations of CMV after preincubation with 20
µM phosphoinositide 3,4-bisphosphate (PI 3,4-P2)or
phosphoinositide 4,5-bisphosphate (PI 4,5-P2) in 0.2 mM
phosphatidylcholine-containing micelles as described in MATERIALS AND
METHODS. B: ATP-dependent transport in CMV isolated from liver
perfused with buffer (open bars), dBcAMP (gray bar), colchicine (closed bar),
or dBcAMP after pretreatment with colchicine (hatched bar). ATP-dependent TC
transport was also measured in the same preparations of CMV after
preincubation with 20 µM PI 3,4-P2 in 0.2 mM
phosphatidylcholine-containing micelles as above. ATP-dependent TC transport
in different preparations of CMV from control animals (n = 6) varied
between 22 and 27 pmol TC · mg protein-1 ·
min-1. Results are means ± SD; n = 3
independent experiments performed in triplicate. See legend of
Fig. 2 regarding statistical
analysis.
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Fig. 6. PI3K activity and ATP-dependent TC transport after addition of PI3K peptide
to CMV in vitro. A: PI3K activity in CMV isolated from control (open
bars), dBcAMP-treated (gray bar), and TC-treated (closed bar) rats. CMV were
incubated with PBS or 20 µM PI3K peptide for 10 min before solubilization.
Results are normalized to phosphatidyl inositol-3 phosphate formed in control
CMV (as in legend of Fig. 3)
and presented as %control values (means ± SD) from 3 independent
experiments performed in duplicate samples. B: ATP-dependent TC
transport in CMV. Results are presented as %control values (as in legend of
Fig. 4) from 3 independent
experiments performed in triplicate samples. See legend of
Fig. 2 regarding statistical
analysis.
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RESULTS
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cAMP induces rapid increase in bile acid secretion that is blocked by
WM and colchicine. The effect of WM and colchicine on cAMP-mediated bile
acid secretion in in situ perfused rat liver was determined. To sustain
initial bile flow, the liver was perfused with Krebs-Ringer-bicarbonate buffer
containing 10 µCi [3H]TC and 10 µM unlabeled TC, which
resulted in a baseline TC secretion of 200230 nmol/3 min within 10 min
(Fig. 1A). Addition of
dBcAMP increased TC secretion up to 10-fold (to
2,000 nmol/3 min), which
became maximal within 2030 min of perfusion and was sustained above
1,500 nmol/3 min for the duration of the experiment. When WM was administered
10 min before treatment with dBcAMP, bile acid secretion was reduced below the
baseline level. Addition of WM 10 or 25 min after perfusion with dBcAMP
reduced bile acid secretion by 5060%
(Fig. 1B). These data
indicate that inhibition of PI3K activity blocks cAMP-induced bile acid
secretion in rat liver.
Pretreatment of rats with colchicine before liver perfusion with dBcAMP
reduced TC secretion by 8090% (Fig.
1C). Perfusion of control rat liver with dBcAMP also
doubled bile flow, which was reduced by 3040% by pretreatment with
colchicine (data not shown). Thus dBcAMP-mediated bile acid secretion and, to
a lesser extent, bile flow require an intact microtubular system.
Effect of WM and colchicine on cAMP-mediated recruitment of
ATP-dependent transporters to the canalicular membrane. We examined the
effect of WM and colchicine on dBcAMP-induced recruitment of bsep and mrp2 to
the canalicular membrane (Fig.
2). CMV were prepared from dBcAMP-perfused liver with or without
inhibitors under the conditions described in MATERIALS AND METHODS.
As previously reported for other ABC canalicular transporters
(11), dBcAMP induced a two- to
threefold increase in canalicular bsep and mrp2 protein content
(Fig. 2A). Perfusion
of liver with WM reduced canalicular bsep by 20% and mrp2 to 50% below the
level detected in control animals (Fig.
2A, bottom). However, WM, given before or after
treatment with dBcAMP, had no effect on cAMP-induced accumulation of bsep or
mrp2 in CMV.
Pretreatment of rats with colchicine reduced mrp2 and bsep content in CMV
below baseline levels as statistically demonstrated by t-test. In
contrast to the lack of effect of WM on cAMP-induced recruitment of ABC
transporters to the canalicular membrane, colchicine decreased dBcAMP-induced
recruitment of mrp2 but had no statistically significant effect on bsep
accumulation in CMV (Fig.
2B).
Effect of dBcAMP, WM, and colchicine on PI3K activity in CMV from in
situ perfused liver. To correlate ATP-dependent TC transport with PI3K
activity, we quantified total PI3K activity in solubilized CMV prepared from
rats treated in situ with dBcAMP with or without WM or colchicine. Perfusion
of the liver with dBcAMP resulted in three- to fourfold increase in total PI3K
activity in CMV (Fig.
3A), which is similar to the previously reported increase
in PI3K activity in CMV in response to TC
(27,
28). WM decreased baseline
PI3K activity to <50% and abolished dBcAMP-mediated increase in PI3K
activity. Similar inhibition of PI3K activity was observed in CMV from
colchicine-treated animals (Fig.
3B).
Effect of dBcAMP on ATP-dependent TC transport in CMV isolated from
liver perfused with WM or colchicine. We compared the activity of
ATP-dependent TC transport in CMV isolated from control and dBcAMP-treated
rats with or without WM and colchicine
(Fig. 4). ATP-dependent
transport of TC in CMV increased 1.5- to 1.7-fold in response to dBcAMP. WM
reduced baseline TC transport in CMV by 70% and abolished dBcAMP-mediated
increase in TC transport (Fig.
4A). Colchicine pretreatment also reduced ATP-dependent
TC transport below baseline and blocked cAMP-mediated increase of TC transport
(Fig. 4B).
Effect of PI3K lipid products on ATP-dependent TC transport in CMV from
rats treated with WM or colchicine. To determine whether addition of PI3K
lipid products restores TC transport in CMV isolated from rats treated with WM
or colchicine, CMV from rats treated in situ were incubated with different
combinations of polyphosphoinositides (Fig.
4). ATP-dependent TC transport was quantified after incubating CMV
with PI 3,4-P2, a product of PI3K, or its isomer, PI
4,5-P2. Addition of PI 3,4-P2 to CMV isolated from
control or dBcAMP-perfused rat increased ATP-dependent TC transport
±1.5-fold, whereas addition of PI 4,5-P2 decreased TC
transport below 30%. Incubation of WM-treated CMV with PI 3,4-P2
but not with PI 4,5-P2 restored ATP-dependent TC transport
(Fig. 4A). Similar
results were obtained when CMV from rats treated with colchicine were
incubated with PI 3,4-P2 (Fig.
4B).
dBcAMP induces phosphotyrosine-independent activation of PI3K.
Activation of p85-associated PI3K has been linked to phosphotyrosine-mediated
signal transduction, which is detectable in anti-phosphotyrosine immune
precipitates (45,
50). In addition, activation
of some receptor-associated heterotrimeric G proteins leads to activation of
p110
PI3K as well as p85-associated p110
subunit
(29). We determined whether
cAMP-mediated activation of PI3K is associated with phosphotyrosine signaling
and which specific subunits of PI3K are involved. Results were compared with
responses observed after TC administration. Using the same amounts of protein,
we quantified PI3K activity in anti-phosphotyrosine, -P110
,
-p110
, and -p110
immune precipitates by using CMV from rats that
had received TC or dBcAMP intravenously 45 min before preparation of CMV.
Anti-phosphotyrosine immunoprecipitable PI3K activity was low and did not
change following administration of dBcAMP or TC
(Fig. 5). A two- to threefold
increase in immunoprecipitable activity was associated with p85 and with
pP110
, -
, and -
subunits of PI3K in CMV from rats treated
with TC. In contrast, administration of dBcAMP resulted in increased anti-p85,
-p110
, and -p110
but not anti-p110
immune precipitates.
These results indicate that activation of PI3K in response to dBcAMP and TC
involves a phosphotyrosine-independent mechanism and that different subunits
of type I PI3K are involved in each process.
Effect of PI3K peptide on PI3K activity and ATP-dependent transport in
CMV from rats treated with dBcAMP or TC. We used a synthetic 10-mer
rhodamine-linked PI3K peptide that activates PI3K in vivo and in vitro
(15,
17,
18,
28). For these experiments,
rats received a single intravenous injection of PBS, 100 µmol dBcAMP, or
100 µmol TC 45 min before preparation of CMV. PI3K activity and
ATP-dependent transport increased two- to fourfold
(Fig. 6). These data are
consistent with activities measured in CMV after perfusion of liver in situ
with dBcAMP described above and with previously reported data from in situ
perfusion with TC (27,
28).
Incubation of CMV with PI3K peptide for 10 min stimulated PI3K activity
2.5-fold and further increased activity in CMV from dBcAMP-treated animals
fourfold. The latter effect may result from dBcAMP-induced recruitment of bsep
to the canalicular membrane. PI3K peptide also enhanced ATP-dependent TC
transport in control vesicles and further increased transport in CMV from
dBcAMP- and TC-treated rats (Fig.
6B).
 |
DISCUSSION
|
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The hydrocholeretic effect of cAMP has been known for a long time and is
attributed to cAMP production primarily in small bile duct cells in response
to secretin and other hormones released in response to food intake
(9). In addition,
administration in vivo of cAMP or its soluble analog dBcAMP enhances
hepatocellular bile acid secretion
(2) comparable with effects
observed following administration of TC
(27,
33) as well as
vesicular-mediated transcytosis
(13). In the present study,
cAMP-induced bile acid secretion was inhibited by perfusion of liver with WM
before or after treatment with dBcAMP or pretreatment of animals with
colchicine. These data indicate that PI3K and an intact microtubular system
are required for cAMP-stimulated bile acid secretion. The effect of WM on bile
acid secretion was not surprising since PI3K lipid products are required for
bile acid transporter function in vitro and in vivo
(27,
28). The addition of the PI3K
lipid product PI 3,4-P2 but not its isomer PI 4,5-P2 to
CMV isolated from rat liver perfused in situ with WM or colchicine restored
ATP-dependent bile acid transport, which confirms the important role of PI3K
activity in the function of bsep.
PI3K and an intact microtubular system were previously reported to
participate in TC- and cAMP-induced intracellular trafficking of ABC
transporters and in recruitment of PI3K regulatory subunit p85 to the
canalicular membrane (11,
22,
27,
28). In the present study,
perfusion of rat liver with dBcAMP resulted in accumulation of bsep and mrp2
in CMV similar to that observed after TC
(11,
27,
28). However, in contrast to
inhibition of TC-induced recruitment of ABC transporters, WM had no
statistically significant effect on cAMP-induced recruitment of bsep or mrp2,
whereas colchicine decreased cAMP-induced recruitment of mrp2 but not bsep
recruitment. Addition of PI3K lipid product PI 3,4,-P2 to CMV
isolated from WM and colchicine-treated rats increased ATP-dependent TC
secretion above control, which confirms that bsep is functionally active in
these vesicles. Furthermore, addition of PI3K peptide to CMV increased PI3K
activity and ATP-dependent TC transport. These results confirm that bsep can
be activated in CMV isolated from WM and colchicine-perfused liver and that
PI3K is required for its function. The mechanism whereby PI3K peptide
increases PI3K activity in hepatocytes is not known; however, studies
performed in fibroblasts suggest that its binding to PI 4,5-P2, a
substrate for PI3K, makes the substrate more available to the enzyme
(15).
Previous results indicated that the effects of cAMP and TC on intracellular
trafficking of canalicular ABC transporters are additive
(11), which led to the
proposal that the two agonists affect different trafficking routes. Results
from the present studies are consistent with this hypothesis. In contrast to
trafficking induced by TC, canalicular mobilization of ABC transporters in
response to cAMP was independent of PI3K. Our results reveal additional
differences between the trafficking routes of bsep and other canalicular ABC
transporters, such as mrp2. cAMP-mediated recruitment of bsep to the
canalicular membrane was unaffected by colchicine, suggesting possible
independence of the microtubular system. The results do not exclude a role for
microtubules, because in WIF-B9 cells nocodazole completely blocked
trafficking of post-Golgi endosomes that contained green fluorescent protein
(GFP)-bsep or GFP-mdr1 to the canalicular membrane (Wakabayashi Y and Arias
IM, unpublished observations; and Ref.
39).
Pulse-labeling studies in intact rats
(20,
22) and image-analysis studies
using GFP-bsep or GFP-mdr1 in WIF-B9 cells (Wakabayashi Y and Arias IM,
unpublished observations; and Ref.
39) demonstrate that ABC
transporters traffic from the Golgi to the canalicular membrane and that bsep
has one or more large intracellular endosomal pools from which it cycles to
and from the canalicular membrane. Mrp2 also trafficks from Golgi to the
canalicular membrane; however, an intracellular pool
(2) for mrp2 has not been
demonstrated. Thus cAMP-mediated recruitment of bsep may involve a novel
unidentified pathway of trafficking in polarized cells, which differs from
PI3K-dependent trafficking, which is induced by TC.
All isoforms of type I PI3K are expressed in rat liver
(5,
45). Type IA PI3K is composed
of a catalytic and a regulatory subunit; p110
, -
, or -
catalytic subunits associate with regulatory subunits p85, p55, or p50. Type
IA PI3K can be activated by a direct interaction of the catalytic subunits
with p21ras or by specific binding of the regulatory subunits to
phosphotyrosyl peptides containing a YXXM motif
(3). Type IB isoform
p110
, which lacks an NH2-terminal p85-binding domain, is
also activated by binding to p21ras and has a putative pleckstrin
homology domain that interacts with phospholipids and binds to G
.
Binding of G-protein
and
subunits to p110 isoforms activates
PI3K (29,
41). Activation of p110
but not p110
has been previously linked to G protein signaling in some
cells, and synergistic activation of P110
/p85 but not p110
/p85 by
a combination of phosphotyrosyl peptide and G
has been reported
(24,
31). Following the observation
that 3'-phosphoinositides can be generated by mitogenic and nonmitogenic
stimuli (3,
23,
40,
44), it has been repeatedly
demonstrated that type I PI3K and its downstream target, Akt, are activated in
response to many signals, including cAMP
(19,
26,
34,
48,
49). Our data and previous
reports demonstrate that PI3K activation in response to cAMP is
phosphotyrosine independent
(19,
34).
Functional differences involving PI3K isoforms occur in some cells.
Activation of p110
has been associated with de novo DNA synthesis,
whereas p110
is implicated in cell survival
(1). The role of individual
isoforms in vesicular trafficking and in intracellular compartmentalization of
PI3K subunits has not been described in polarized cells. In the present
studies, cAMP-stimulated PI3K activity was associated with p110
and
-
but not p110
isoform, and these effects were not associated
with tyrosine phosphorylation of intracellular proteins. These results suggest
a coordinate effect of cAMP on activation of types IA and IB PI3K that is
independent of tyrosine phosphorylation and, probably, p21ras.
Many effects of cAMP are PKA independent, and conflicting data have been
generated based on use of PKA inhibitors
(6,
19,
48). Discovery of a novel cAMP
receptor, Epac, which is a cAMP-activated Rap1 guanine-nucleotide exchange
factor, provides a new opportunity to elucidate cAMP-mediated cell signaling
(8,
10). Many cAMP effects, which
were previously attributed to PKA, may require Epac/Rap1. cAMP-mediated
activation of Epac and PKA has opposite effects on Akt activation
(26), which has been
attributed to their different intracellular localizations
(36). The role of Rap 1 and
Epac in cAMP-mediated vesicular trafficking in polarized cells is the subject
of our current studies.
In summary, dBcAMP and TC-induced bile secretion in rat liver involves
different pathways. In contrast to effects following TC administration,
cAMP-stimulated intracellular trafficking of bsep and mrp2 is PI3K
independent. These observations prompt revision of current concepts regarding
the role of cAMP and PI3K in intracellular trafficking, regulation of
canalicular ABC transporters, and bile secretion.
 |
DISCLOSURES
|
---|
This research was supported, in part, by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-54785 (to I. M. Arias), DK-35652 (to
I. M. Arias and L. Varticovski), and 30-DK-34928 (Digestive Disease Center),
and National Cancer Institute Grant CA-53094 (to L. Varticovski).
 |
ACKNOWLEDGMENTS
|
---|
We thank P. Ujhazy for assistance with Western blot analysis.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: I. M. Arias, Dept. of
Physiology, Tufts Univ. School of Medicine, 136 Harrison Ave., M&V7,
Boston, MA 02111 (E-mail:
iarias{at}helix.nih.gov).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
 |
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