From the Tufts University School of Medicine, Department of Physiology, Boston, Massachusetts 02111
Received for publication, August 25, 2000, and in revised form, December 1, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ABC transporter trafficking in rat
liver induced by cAMP or taurocholate and
[35S]methionine metabolic labeling followed by
subcellular fractionation were used to identify and characterize
intrahepatic pools of ABC transporters. ABC transporter trafficking
induced by cAMP or taurocholate is a physiologic response to a temporal
demand for increased bile secretion. Administration of cAMP or
taurocholate to rats increased amounts of SPGP, MDR1, and MDR2 in the
bile canalicular membrane by 3-fold; these effects abated after 6 h and were insensitive to prior treatment of rats with cycloheximide.
Half-lives of ABC transporters were 5 days, which suggests cycling of
ABC transporters between canalicular membrane and intrahepatic sites
before degradation. In vivo [35S]methionine
labeling of rats followed by immunoprecipitation of (sister of
P-glycoprotein) (SPGP) from subcellular liver fractions revealed a
steady state distribution after 20 h of SPGP between canalicular
membrane and a combined endosomal fraction. After mobilization of
transporters from intrahepatic sites with cAMP or taurocholate, a
significant increase in the amount of ABC transporters in canalicular
membrane vesicles was observed, whereas the decrease in the combined
endosomal fraction remained below detection limits in Western blots.
This observation is in accordance with relatively large intracellular
ABC transporter pools compared with the amount present in the bile
canalicular membrane. Furthermore, trafficking of newly synthesized
SPGP through intrahepatic sites was accelerated by additional
administration of cAMP but not by taurocholate, indicating two distinct
intrahepatic pools. Our data indicate that ABC transporters cycle
between the bile canaliculus and at least two large intrahepatic ABC
transporter pools, one of which is mobilized to the canalicular
membrane by cAMP and the other, by taurocholate. In parallel to
regulation of other membrane transporters, we propose that the
"cAMP-pool" in hepatocytes corresponds to a recycling
endosome, whereas recruitment from the "taurocholate-pool" involves
a hepatocyte-specific mechanism.
The bile canalicular membrane of the mammalian hepatocyte contains
several primary active transporters that couple ATP hydrolysis to the
transport of specific substrates into the bile canaliculus (1-4).
These transporters are members of the superfamily of ATP binding cassette
(ABC)1 membrane transport
proteins (5) and currently include P-glycoprotein (MDR1) for organic
cations (6), MDR2 for phosphatidylcholine translocation (7, 8), sister
of P-glycoprotein (SPGP), the canalicular bile salt export pump (BSEP)
(9), and MRP2 for non-bile acid organic anions (10).
Recent studies indicate that the amount of each ABC transporter in the
canalicular membrane is regulated by the physiological demand to
secrete bile acids. Intravenous administration to rats of
dibutyryl-cAMP (Bt2cAMP) or taurocholate (TC)
rapidly and selectively increased the functional activity and amount of
each ABC transporter in the canalicular membrane; these effects were
inhibited by prior administration of colchicine, which disrupts
microtubules (11), and Wortmannin, which inhibits phosphatidylinositol
3-kinase (12). These observations indicate that an intracellular
microtubule-dependent transport mechanism, which is
sensitive to active phosphatidylinositol 3-kinase, is required for
trafficking of ABC transporters to the canalicular membrane.
ATP-dependent transport activity of SPGP and MRP2 within
the bile canalicular membrane requires active phosphatidylinositol
3-kinase and is regulated by 3'-phosphorinositide products of
phosphatidylinositol 3-kinase (13).
ABC transporters are essential for biliary secretion in mammalian
liver, and their amounts and activities in the canalicular membrane are
tightly regulated. Inherited defects in SPGP and MRP2 proteins result
in familial intrahepatic cholestasis (for review, see Ref. 14).
Therefore, defects in trafficking and regulation of ABC transporters
may result in insufficient amounts or activity of ABC transporters in
the bile canalicular membrane, the consequences of which are impaired
bile secretion and cholestasis (15).
The increase in the content of ABC transporters in the canalicular
membrane after administration of Bt2cAMP or TC in
vivo (11) and in isolated perfused rat liver (12, 13) occurred within minutes after the administration of the effectors. These observations suggest that the increment in canalicular ABC transporters is likely to represent recruitment from pre-existing intrahepatic pools
rather than from enhanced transcription or translation. Furthermore,
the simultaneous administration of Bt2cAMP and TC resulted
in additive rather than alternative effects (11). These observations
suggest that Bt2cAMP and TC recruit ABC transporters to the
canalicular membrane from different intrahepatic pools. Moreover,
investigations of Golgi-to-bile canaliculus pathways of newly
synthesized ABC transporters (16, 17) also suggest that SPGP, in
particular, is sequestered in intrahepatic pools before reaching the
bile canalicular plasma membrane. The present report concerns
biochemical identification and characterization of these intrahepatic
ABC transporter pools and identification of possible mechanisms by
which ABC transporters are made available to the canalicular membrane.
Materials--
Expre35S35S protein label
was supplied by PerkinElmer Life Sciences. All other chemicals
were of highest purity available and were purchased from Sigma.
Monoclonal antibody C219 (anti-MDR1/MDR2) was from Centocor (Malvern,
PA). Polyclonal anti-SPGP antibody LVT90 was developed in our
laboratory (15). Polyclonal anti-cCAM105 antibody was a gift from
S. H. Lin (Houston, TX). Antibodies against Rab proteins were from
Santa Cruz Biotechnology.
Miscellaneous Methods--
Young adult male Harlan
Sprague-Dawley rats (300-350 g) kept on a standard diet were used for
preparation of liver subcellular fractions. In some experiments, rats
were pretreated by intravenous injection with Bt2cAMP (20 µmol/kg) or TC (50 µmol/kg) dissolved in 1 ml of PBS for the
indicated time periods. In control experiments, rats received 1 ml of
PBS. Further experimental details are stated in the figure legends.
Preparation and characteristics of rat liver canalicular membrane
vesicles (CMV), sinusoidal/basolateral membrane vesicles (SMV), and
Golgi membranes were described previously (16, 18, 19). A combined
endosomal fraction (CEF) from rat liver was prepared using the protocol
of Khan et al. (20). Metabolic [35S]methionine
labeling and immunoprecipitation from subcellular rat liver fractions
were performed as described earlier (16). Proteins were separated by
SDS-PAGE according to Laemmli (21). For Western blotting, polypeptides
were electrotransferred (22) on nitrocellulose membranes (Schleicher & Schuell) or polyvinylidene difluoride membrane (Milipore) for the Rab
proteins. Antibodies were detected by incubation with horseradish
peroxidase-conjugated secondary antibodies followed by detection with
an enhanced chemiluminescence system from PerkinElmer Life Sciences.
The method of Lowry et al. (23) was used for protein
measurements with bovine serum albumin as standard.
Bt2cAMP and TC Transiently Increase the Canalicular
Amount of ABC Transporters--
Gatmaitan et al. (11)
demonstrate that intravenous injection of rats with Bt2cAMP
or TC increased the amount of canalicular proteins in the bile
canalicular membrane. To determine the duration and magnitude of the
response to Bt2cAMP and TC administration, we investigated
the time courses of these effects. Groups of rats were injected with
either Bt2cAMP (20 µmol/kg) or TC (50 µmol/kg) for
various time periods, and canalicular amounts of ABC transporters were
determined in CMVs by Western blotting (Fig.
1). We demonstrated earlier that
monoclonal C219 antibody is specific for rat MDR1 and MDR2 and that
polyclonal LVT90 antibody is specific for SPGP (16). After treatment
with Bt2cAMP, an increase in MDR1, MDR2, and SPGP in CMVs
was observed after 15 min; the effect peaked at 45 min to 4 h and
disappeared after 6 h. TC treatment resulted in a significant
increase in MDR1, MDR2, and SPGP after 15 min; the effect peaked at 45 to 90 min and then declined to the basal level after 4 h. From
three sets of independent experiments, the maximal increase in the
amount of ABC transporters in the canalicular membrane was 3.6 ± 0.9-fold after stimulation with Bt2cAMP and 2.7 ± 0.7-fold after treatment with TC.
Onset and duration of the Bt2cAMP and TC effects probably
depend on the dose of the injected drug, which has not been
investigated in detail. However, the increase in ABC transporter
amounts in the bile canaliculus caused by Bt2cAMP or TC is
transient, which prompts two questions: Where do the additional ABC
transporters originate from? Where do they go after the effects have abated?
TC and cAMP Effects Are Independent of de Novo Protein
Synthesis--
Additional ABC transporters in the bile canaliculus
upon stimulation with Bt2cAMP or TC could theoretically
result from enhanced protein biosynthesis and/or recruitment of
transporters from pre-existing intracellular pools. To discriminate
between these possibilities, we determined whether the increase in ABC
transporters in the bile canaliculus remained when protein synthesis
was inhibited. Protein biosynthesis was efficiently blocked by
intraperitoneal injection of rats with cycloheximide (5 mg/kg) for 30 min. Pretreatment with cycloheximide completely abolished metabolic
labeling with [35S]methionine of proteins in CMVs. This
was demonstrated by detection of 35S-labeled proteins in
CMVs with a scintillation counter and of radiolabeled proteins using
PhosphorImager after separation of CMV proteins by SDS-PAGE
(Fig. 2).
Rats were injected intraperitonally with cycloheximide for 30 min
followed by intravenous injection of either Bt2cAMP or TC for 1 h. CMVs were then prepared, and proteins from rat liver homogenate and CMVs were separated by SDS-PAGE. The amount of ABC
transporters in both preparations was quantified by Western blotting
with C219 (MDR1, MDR2) and LVT90 (SPGP) antibodies and compared with
control experiments (Fig. 3).
Cycloheximide inhibits protein biosynthesis; however, in some instances
cycloheximide can also cause superinduction of proteins (24, 25). In
rat liver homogenate no significant change in the amount of ABC
transporters was observed after pretreatment of rats with
cycloheximide, Bt2cAMP, and TC in various combinations (Figs. 3, A and B). These data indicate that
superinduction of proteins by cycloheximide is not a concern in our
study regarding ABC transporters. The total amount of SPGP, MDR1, and
MDR2 in rat liver remained constant after administration of
cycloheximide, Bt2cAMP, and TC.
Pretreatment of rats with cycloheximide for 30 min had no effect on the
basal protein amount of MDR1, MDR2, and SPGP in CMVs. Cycloheximide
pretreatment did not reduce the increase in ABC transporters in the
bile canaliculus in response to Bt2cAMP or TC. Regardless
of prior cycloheximide administration, Bt2cAMP or TC
significantly increased the canalicular content of MDR1, MDR2, and SPGP
when compared with control animals (Figs. 3, C and
D). These experiments demonstrate that additional ABC
transporters in the bile canalicular membrane after stimulation with
Bt2cAMP or TC do not result from enhanced translation but
from a redistribution between intrahepatic ABC transporter pools and
the bile canalicular membrane.
Half-lives of Canalicular ABC Transporters--
Where do the
canalicular ABC transporters go after peak stimulation by
Bt2cAMP or TC and their amounts in the bile canalicular membrane return to basal levels? Two possible scenarios were considered as follows. ABC transporters either return to intrahepatic sites, or in
parallel with other canalicular protein receptors, they are degraded.
The latter presumably should be manifested by relatively short
half-lives for canalicular ABC transporters. Therefore, we determined
the half-lives of the canalicular ABC transporters and compared the
results to previously established half-lives of other hepatocyte plasma
membrane proteins.
A group of rats was labeled by intravenous injection with
[35S]methionine for 1, 3, 5, 8, and 10 days (without
chase), and the content of newly synthesized MDR1, MDR2, and SPGP was
determined by immunoprecipitation from CMVs and homogenate. The
half-life of cCAM105 was also determined as a control. Protein
half-lives were determined from semi-logarithmic plots of
35S intensity versus labeling time (Fig.
4). As observed earlier (16), MDR1 and
MDR2 could not be immunoprecipitated with C219 antibody from liver
homogenate, probably due to low abundance of the antigens.
Assuming first order decay, cCAM105 had an apparent half-life of 5 days
as measured in homogenates and CMVs, which corresponds to an earlier
published report (26). A 5-day half-life was also determined for MDR1
and MDR2 in CMVs. For SPGP, a half-life of 4 days was measured from
CMVs and 6 days from homogenate. It is not clear why the apparent
half-lives differ when determined from the two preparations. However,
it has been observed before that the apparent half-lives of hepatic
proteins are shorter when determined from plasma membranes as compared
with results in homogenates (26).
The half-lives of several hepatocyte plasma membrane proteins are
presented in Table I and were
obtained from metabolic studies using radiolabeled amino acids. Except
for nucleotide pyrophosphatase and the polymeric IgA receptor, which
have short half-lives, all other hepatic plasma membrane proteins
previously investigated have half-lives of 2-9 days. The short
half-life of the polymeric IgA receptor is accounted for by extensive
loss into the bile (26). Since it is difficult to prevent long term
label reincorporation in vivo (26, 29), the data probably
represent upper limits. Similar to other hepatic plasma membrane
proteins (Table I), canalicular SPGP, MDR1, and MDR2 have relatively
long half-lives (t1/2 > 1 day) and are likely to undergo lysosomal degradation (31).
Trafficking of ABC transporters from intrahepatic sites to the bile
canalicular membrane is physiologic and results from increased need to
secrete bile. Therefore, we postulate that canalicular ABC transporters
cycle between the apical membrane and intrahepatic sites prior to
undergoing degradation.
Large Amounts of SPGP Reside in Intracellular Pools--
A CEF
from rat liver was prepared (20) to investigate the dynamics and steady
state levels of SPGP in intrahepatic pools. CEFs, prepared by
centrifugation of a microsomal fraction of rat liver on a discontinuous
sucrose gradient, are highly enriched in endosomal markers Rab5 (early
endosomes) and Rab11 (apical recycling endosomes) (32) as compared with
homogenates and CMV and SMV plasma membrane subfractions (Fig.
5).
In a recent paper (16), we described trafficking of newly synthesized
SPGP by investigating the kinetics of
[35S]methionine-labeled SPGP in liver homogenate, Golgi
membranes, CMVs, and SMVs. SPGP was never observed in SMVs, indicating
nontranscytotic targeting. Furthermore, in study of SPGP trafficking,
there was a 1.5-h lag between passage through Golgi and arrival at the
canalicular membrane, which suggests transient sequestration in
intrahepatic sites. We expanded this experiment to include CEF.
Trafficking of newly synthesized SPGP after pulse-chase labeling with
[35S]methionine through Golgi membranes, CEF, and CMVs is
shown in Fig. 6.
Radiolabeled SPGP peaked in Golgi membranes after a chase time of 30 min and thereafter was virtually absent from the Golgi, indicating that
processing and passage of SPGP through Golgi is complete after 30-60
min. SPGP peaked in CEF at 1 h and, after a chase time of 2 h, first appeared in CMVs. These experiments demonstrate that newly
synthesized SPGP is targeted through an endosomal compartment before
reaching the bile canalicular membrane. Furthermore, SPGP is not
completely transferred from CEF, and a significant amount of SPGP
remains in the endosomal fraction. These results suggest a distribution
of SPGP between canalicular membrane and intracellular pools (2- and
3-h chase). This was also observed after a chase time of 20 h,
which presumably represents steady state distribution of SPGP between
the bile canaliculus and intracellular pools under basal conditions.
Immunoblots were used to determine the distribution of SPGP and
MDR1/MDR2 between CMVs and CEF under steady state conditions and
after induction of trafficking with Bt2cAMP or TC (Fig.
7). Administration to rats of
Bt2cAMP or TC for 1 h significantly increased the
amounts of SPGP and MDR1/MDR2 in the canalicular membrane, whereas the
amounts in liver homogenate remained constant. These data suggest a
redistribution of existing ABC transporters between intracellular pools
and the canalicular membrane upon stimulation with Bt2cAMP
and TC. Surprisingly, no decrease in the ABC transporter amount could
be detected in CEF; the amount of SPGP, MDR1, and MDR2 remained
constant after stimulation with Bt2cAMP and TC. These
observations can be best explained by a large intrahepatic pool of ABC
transporters, only a small portion of which is present in the bile
canalicular membrane. In this scenario, a small portion of ABC
transporters trafficking from a large endosomal pool to a small bile
canalicular pool results in significantly increased amount of ABC
transporters in CMVs, whereas any decrease in ABC transporter amount in
CEF remained below the detection limit.
Targeting of Newly Synthesized SPGP to the Bile Canaliculus Is
Accelerated by Bt2cAMP But Not by TC--
Newly
synthesized SPGP is targeted through intrahepatic sites before reaching
the bile canalicular membrane. This was concluded from the 1.5-h lag
time between passage through Golgi and arrival at the hepatocyte apical
pole (16) and was confirmed by [35S]methionine metabolic
pulse-chase labeling of a CEF from rat liver (Fig. 6). The pattern of
ABC transporter trafficking after injection of rats with
Bt2cAMP or TC also suggests the existence of intrahepatic
pools. The two approaches were combined to determine whether
intrahepatic SPGP pools, in which newly synthesized SPGP is sequestered
before reaching the bile canaliculus, is also mobilized by
Bt2cAMP and/or TC. Typically [35S]SPGP
is first detected in CMVs after 2 h in metabolic labeling studies.
Therefore, we determined whether [35S]SPGP can be
detected in CMVs after 1 h of labeling after additional administration of Bt2cAMP or TC (Fig.
8).
As demonstrated before, treatment of rats with Bt2cAMP or
TC for 30 min significantly increased the steady state amount of SPGP
in CMVs. However, accelerated trafficking of newly synthesized SPGP to
the bile canalicular membrane after 1 h was observed only when
rats were pretreated with Bt2cAMP. Pretreatment with TC did not accelerate Golgi-to-bile canaliculus trafficking of newly synthesized SPGP. Altered schedules for TC administration 15 or 45 min
after metabolic labeling also did not accelerate
[35S]SPGP trafficking to the canalicular membrane (data
not shown). Thus, trafficking of newly synthesized SPGP through
intrahepatic pools to the bile canalicular membrane was accelerated by
cAMP but not by TC.
Previous morphological studies in rats rendered cholestatic by
bile duct ligation (33), phalloidin (34), or lipopolysaccharide (35)
suggest that MRP2 and SPGP may traffic from the bile canaliculus to
intracellular sites. In addition, MRP2 (35) and SPGP (9) were observed
by immunogold staining and electron microscopy in undefined vesicular
structures that were distinct from the bile canaliculus.
The present study demonstrates that selective increase in ABC
transporters in the canalicular membrane after stimulation with cAMP or
TC is independent of protein de novo synthesis, indicating that additional transporters, upon stimulation, are recruited from
pre-existing intrahepatic pools. Stimulation by the second messenger,
cAMP, and the SPGP substrate, TC, probably mimic naturally occurring
processes. The cAMP and TC effects are transient, and the half-lives of
SPGP, MDR1, and MDR2 are not significantly shorter than those of other
hepatic plasma membrane proteins, which suggests that ABC transporters
cycle between intrahepatic pools and the canalicular membrane. The
steady state distribution of SPGP between intrahepatic pools and
canalicular membrane is probably regulated by cAMP and TC depending on
the physiological demand to secrete bile.
Previous studies (11) indicate that the increasing effects of
Bt2cAMP and TC in bile canalicular ABC transporter amount are additive rather than alternative, which suggests the presence of at
least two distinct intrahepatic pools of ABC transporters, one of which
is mobilized to the canalicular membrane by Bt2cAMP (cAMP
pool) and the other by TC (TC-pool). We here demonstrate that targeting
of newly synthesized SPGP through intrahepatic sites to the bile
canalicular membrane is accelerated by Bt2cAMP but not by
TC. This observation supports the hypothesis of two distinct
intrahepatic pools of SPGP. After passage through Golgi, SPGP
accumulates in an intrahepatic cAMP pool and later equilibrates with
the TC pool. Whether equilibration of newly synthesized SPGP with the
TC pool occurs from the bile canalicular membrane or the cAMP pool
remains unclear. A tentative model for intrahepatic pathways of ABC
transporters is shown in Fig. 9. Newly
synthesized MDR1 and MDR2 bypass the intracellular pools on their
journey to the bile canaliculus (16, 17). However, at steady state levels, MDR1 and MDR2 are also mobilized to the bile canalicular membrane by Bt2cAMP and TC, indicating that these ABC
transporters also equilibrate with intrahepatic pools after reaching
the bile canalicular membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (28K):
[in a new window]
Fig. 1.
Bt2cAMP and TC temporarily
increase canalicular amount of ABC transporters. Groups of rats
were injected with Bt2cAMP (20 µmol/kg) or TC (50 µmol/kg) in PBS, and livers were excised after the indicated time
points for the preparation of CMVs. Equal amounts of CMV proteins (20 µg/lane) were separated by SDS-PAGE, blotted onto nitrocellulose
membrane, and probed with LVT90 (SPGP) and C219 (MDR1/MDR2) antibodies.
A control group of rats received only injection with PBS. Panels
A and C show representative results observed in three
independent sets of rats. Panels B and D show
densitometric quantitations of Western blots from three independent
sets of rats (mean values ± S.D., n = 3) with TC
(mean values ± S.D., n = 3). The
asterisk indicates values significantly different from
control using Student's t test (p < 0.05).
View larger version (35K):
[in a new window]
Fig. 2.
Cycloheximide efficiently inhibits protein
biosynthesis. To establish the effectiveness of cycloheximide
(CHX) in blocking protein biosynthesis in vivo, a
rat was treated with an intraperitoneal injection of cycloheximide (5 mg/kg) for 30 min before metabolic labeling with
[35S]methionine for 2 h. CMVs were prepared, and
radioactivity was determined from an aliquot by liquid
scintillation counting (A). Furthermore, an aliquot of CMVs
was separated by SDS-PAGE and stained with Coomassie (B),
and radioactivity was then detected by phosphorimaging (C).
The results were compared with CMVs from a rat without cycloheximide
pretreatment. The results shown were established in a single
experiment; error bars indicate S.D. from three
replica.
View larger version (36K):
[in a new window]
Fig. 3.
TC and cAMP effects are independent of
de novo protein synthesis. Rats were injected
intraperitonally with cycloheximide (CHX) (5 mg/kg) for 30 min followed by intravenous injection of either Bt2cAMP (20 µmol/kg) or TC (50 µmol/kg) for 1 h. In control experiments,
rats received an injection with PBS. Equal amounts of homogenate
(HOM, A and B) (50 µg/lane) and CMVs (C and D) (20 µg/lane) were separated by SDS-PAGE, blotted onto
nitrocellulose membrane, and probed with LVT90 (SPGP) and C219
(MDR1/MDR2) antibodies. Panels A and C show
representative results observed in three independent sets of rats.
Panels B and D show densitometric quantitations
of Western blots from three independent sets of rats (mean values ± S.D., n = 3). The asterisk indicates
values significantly different from control using Student's
t test (p < 0.05).
View larger version (38K):
[in a new window]
Fig. 4.
Half-lives of canalicular proteins. Rats
were metabolically labeled by intravenous injection with
[35S]methionine (5 mCi) for 1, 3, 5, 8, and 10 days.
cCAM105 (A) and MDR1/MDR2 and SPGP (C) were then
immunoprecipitated from liver homogenate (HOM) and CMVs.
Immunoprecipitates were separated by SDS-PAGE, and radiolabeled bands
were detected and quantified by phosphorimaging. Half-lives of the
proteins were estimated from semi-logarithmic plots (B and
D) of the relative 35S intensity (highest values
equal 100) versus labeling time. Assuming first order decay,
data points were fitted linearly by the least square method. Determined
half-lives are: cCAM105, 5 days; MDR1 and MDR2, 5 days; SPGP, 4 days
(CMVs) and 6 days (HOM). The half-lives were established with one set
of five rats.
Half-lives of several hepatocyte plasma membrane proteins
View larger version (33K):
[in a new window]
Fig. 5.
Rab 5 and Rab 11 are highly enriched in a CEF
from rat liver. Rat liver homogenate (HOM), CEF, CMVs,
and SMVs were separated by SDS-PAGE (10 µg of protein of each
fraction), blotted onto a polyvinylidene difluoride membrane, and
probed with anti-Rab5 (early endosomes) and anti-Rab11 (apical
recycling endosomes) antibodies.
View larger version (36K):
[in a new window]
Fig. 6.
Newly synthesized SPGP is targeted through an
endosomal compartment. Rats were pulse-labeled for 15 min with
[35S]methionine (5 mCi) and then chased with unlabeled
methionine for 15 and 30 min and 1, 2, 3, and 20 h, respectively.
SPGP was then immunoprecipitated from Golgi membranes, CEF, and CMVs.
Immunoprecipitates were separated by SDS-PAGE, and
[35S]SPGP was detected by phosphorimaging. Panel
A shows representative results observed in three independent sets
of rats; arrowheads indicate the position of mature
antigens. To establish the kinetics of newly synthesized SPGP
trafficking through cellular compartments (squares, Golgi;
diamonds, CEF; circles, CMV), intensities of
[35S]SPGP bands were quantified by phosphorimaging. The
relative intensity (highest reading in each fraction equals 100) was
plotted versus labeling time (B); mean
values ± S.D., n = 3.
View larger version (51K):
[in a new window]
Fig. 7.
Under basal conditions the major amount of
ABC transporters resides in intrahepatic pools. Rats were injected
with Bt2cAMP (20 µmol/kg) or TC (50 µmol/kg), and
livers were excised after 1 h for the preparation of CMVs and CEF.
A control group of rats received only injection with PBS. Equal amounts
of homogenate (HOM) 50 µg/lane), CMVs (20 µg/lane), and CEF proteins (20 µg/lane) were
separated by SDS-PAGE, blotted onto nitrocellulose, and probed with
LVT90 (SPGP) and C219 (MDR1/MDR2) antibodies. Panels A and
C show representative results observed in three independent
sets of rats. WB, Western blot. Panels B and
D show densitometric quantitations of Western blots from
three independent sets of rats (mean values ± S.D.,
n = 3). The asterisk indicates values
significantly different from control using Student's t test
(p < 0.05).
View larger version (30K):
[in a new window]
Fig. 8.
Membrane targeting of newly synthesized SPGP
is accelerated by cAMP but not by TC. Rats were pulse-labeled for
15 min with [35S]methionine (5 mCi) and then chased with
unlabeled methionine for 1 h. At a chase time of 30 min, rats
received an intravenous injection of PBS (control), Bt2cAMP
(20 µmol/kg), or TC (50 µmol/kg). CMVs were prepared from the rat
livers from which SPGP was immunoprecipitated. A,
immunoprecipitates (IP) were separated by SDS-PAGE and
[35S]SPGP detected by phosphorimaging (a typical result,
observed in three independent sets of three rats is shown).
B, equal protein amounts (20 µg) of the same CMV
preparations were separated by SDS-PAGE, blotted onto nitrocellulose
membrane, and probed with anti-SPGP antibody. WB, Western
blot. C, Western blots from three independent sets were
quantitated with a laser densitometer and compiled; mean values ± S.D., n = 3. The asterisk indicates values
significantly different from control using Student's t test
(p < 0.05).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 9.
Tentative model for ABC transporter
trafficking in rat hepatocytes. Dashed arrows indicate
possible pathways of ABC transporters from the TC pool. See text for
explanation.
Using metabolic pulse-chase [35S]methionine labeling, a CEF was identified to be the site at which newly synthesized SPGP accumulates before being targeted to the bile canalicular membrane. After newly synthesized SPGP reached the bile canalicular membrane, a significant amount remained in CEF 20 h after labeling, suggesting steady state distribution of SPGP between canalicular membrane and intrahepatic pools. Since absolute amounts of ABC transporters in the prepared membrane fractions are not known nor how much of the total amount in the rat hepatocyte they represent, precise calculation of the intrahepatic/canalicular ratio exceeds the limits of these experiments. For MRP2, this ratio has been calculated to be ~1:1 by quantitation of immunogold-stained MRP2 in electron microscopy (35) in rat liver under basal conditions. Investigation of ABC transporters by immunoblots at steady state levels revealed that, after stimulation with Bt2cAMP or TC, the amounts of MDR1, MDR2, and SPGP significantly increased in CMVs, but no change was observed in CEF prepared from the same rat liver. This observation is in accordance with the presence of "large" intrahepatic pools. Under basal conditions, most MDR1, MDR2, and SPGP appears to reside in intrahepatic pools rather than in the canalicular membrane. However, the increase in the amount of ABC transporter in CMVs after stimulation with Bt2cAMP or TC is ~3-fold for each effector. Taking into account that Bt2cAMP and TC recruit transporters from different intracellular sources, the intrahepatic pool of ABC transporters is at least 6-times more than the amount present in the bile canalicular membrane. Since this calculation presumes that all intracellular ABC proteins are translocated to the bile canalicular membrane upon stimulation, this number represents a lower limit. Thus, the intrahepatic/canalicular ratio of MDR1, MDR2, and SPGP probably exceeds 6:1.
Upon stimulation with cAMP, trafficking of membrane transporters from intracellular sites to the plasma membrane has been described in several systems, i.e. (i) cystic fibrosis transmembrane regulator (CFTR) channel into the apical surface of rat duodenal villous epithelia (36); (ii) sodium taurocholate cotransport protein (ntcp) in the basolateral membrane of rat hepatocytes (37); (iii) aquaporin-2 water channel into the apical membrane of LLC-PK1 cells, a polarized renal cell line (38); (iv) H+/K+-ATPase into the apical membrane of gastric parietal cells (39); (v) insulin-responsive glucose transporter 4 into the plasma membrane of rat adipocytes (40). In each of these examples, recruitment of transporters to the plasma membrane from a recycling endosome has been suggested. In particular, trafficking of glucose transporter 4 in rat adipocytes has parallels to that of ABC transporter trafficking in rat hepatocytes. Glucose transporter 4 traffics from distinct intracellular sites to the plasma membrane in response to cAMP and insulin (40). In analogy to these other systems, we propose that cAMP recruits ABC transporters to the bile canalicular membrane from a recycling endosome, whereas the effect of TC appears to be hepatocyte-specific and involves a different mechanism.
The present study demonstrates that a substantial portion of
canalicular ABC transporters resides in intracellular pools in hepatocytes and that the transporters can be transiently recruited from
different intrahepatic pools to the bile canalicular membrane in
response to cAMP and TC. Since ABC transporters are critical for bile
formation, the present studies prompt revision of current concepts of
bile secretion and raise a question with regard to the mechanism. Do
intrahepatic pools of ABC transporters supply additional transporters
to the bile canaliculus only to cope with temporarily higher metabolic
demand to secrete bile, or are these intracellular ABC transporter
pools part of an unidentified more sophisticated bile secretion
mechanism? The latter possibility is supported by the finding that
intrahepatic pools of MRP2, which also colocalize with SPGP, secrete a
MRP2 substrate into intracellular structures (33). A further challenge
is the correlation of biochemically observed intrahepatic ABC
transporter pools with morphological structures. In this regard,
studies in Wif-B cells, a tissue culture model for functionally active,
polarized hepatocytes (17, 41, 42), will be a useful tool. Wif-B cells
infected with an adenoviral construct containing SPGP tagged with
enhanced yellow fluorescent protein showed cycling of SPGP between the
canalicular membrane and an intracellular compartment that colocalized
with anti-Rab11 (recycling endosomes) antibody immunostaining (43).
This project is presently being pursued in our laboratory.
![]() |
FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Research Grant Ki 640 (to H. K.) and by National Institutes of Health Grants DK35652 (NIDDK) and 30DK34928 (Digestive Disease Center, NIDDK) (to I. M. A.).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.
Present address: Max-Planck-Institut für molekulare
Physiologie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.
§ To whom correspondence should be addressed: Tufts University School of Medicine, Dept. of Physiology, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6739; Fax: 617-636-0445; E-mail: irwin. arias{at}tufts.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M007794200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ABC, ATP binding cassette; Bt2cAMP, dibutyryl cyclic AMP; CEF, combined endosomal fraction; CMV, canalicular membrane vesicle; SMV, sinusoidal membrane vesicle; TC, taurocholate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SPGP, sister of P-glycoprotein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Nishida, T., Gatmaitan, Z., Che, M., and Arias, I. M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6590-6594[Abstract] |
2. |
Nishida, T.,
Hardenbrook, C.,
Gatmaitan, Z.,
and Arias, I. M.
(1992)
Am. J. Physiol.
262,
G629-G635 |
3. | Stieger, B., O'Neill, B., and Meier, P. J. (1992) Biochem. J. 284, 67-74[Medline] [Order article via Infotrieve] |
4. |
Gatmaitan, Z. C.,
and Arias, I. M.
(1995)
Physiol. Rev.
75,
261-275 |
5. | Higgins, C. F. (1992) Annu. Rev. Cell Biol. 8, 67-113[CrossRef] |
6. |
Kamimoto, Y.,
Gatmaitan, Z.,
Hsu, J.,
and Arias, I. M.
(1989)
J. Biol. Chem.
264,
11693-11698 |
7. | Ruetz, S., and Gros, P. (1994) Cell 77, 1071-1081[Medline] [Order article via Infotrieve] |
8. | Nies, A. T., Gatmaitan, Z., and Arias, I. M. (1996) J. Lipid Res. 37, 1125-1136[Abstract] |
9. |
Gerloff, T.,
Stieger, B.,
Hagenbuch, B.,
Madon, J.,
Landmann, L.,
Roth, J.,
Hofmann, A. F.,
and Meier, P. J.
(1998)
J. Biol. Chem.
273,
10046-10050 |
10. |
Büchler, M.,
König, J.,
Brom, M.,
Kartenbeck, J.,
Spring, H.,
Horie, T.,
and Keppler, D.
(1996)
J. Biol. Chem.
271,
15091-15098 |
11. |
Gatmaitan, Z. C.,
Nies, A. T.,
and Arias, I. M.
(1997)
Am. J. Physiol.
272,
G1041-G1049 |
12. |
Misra, S.,
Ujhazy, P.,
Gatmaitan, Z.,
Varticovski, L.,
and Arias, I. M.
(1998)
J. Biol. Chem.
273,
26638-26644 |
13. |
Misra, S.,
Ujhazy, P.,
Varticovski, L.,
and Arias, I. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
5814-5819 |
14. | Kullak-Ublick, G. A., Beuers, U., and Paumgartner, G. (2000) J. Hepatol. 32, 3-18[CrossRef][Medline] [Order article via Infotrieve] |
15. | Kipp, H., and Arias, I. M. (2000) Semin. Liver Dis. 20, 339-351[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Kipp, H,
and Arias, I. M.
(2000)
J. Biol. Chem.
275,
15917-15925 |
17. |
Sai, Y,
Nies, A. T.,
and Arias, I. M.
(1999)
J. Cell Sci.
112,
4535-4545 |
18. |
Inoue, M.,
Kinne, R.,
Tran, T.,
Biempica, L.,
and Arias, I. M.
(1983)
J. Biol. Chem.
258,
5183-5188 |
19. | Inoue, M., Kinne, R., Tran, T., and Arias, I. M. (1982) Hepatology 2, 572-579[Medline] [Order article via Infotrieve] |
20. |
Khan, M. N.,
Baquiran, G.,
Brule, C.,
Burgess, J.,
Foster, B.,
Bergeron, J. J. M.,
and Posner, B. I.
(1989)
J. Biol. Chem.
264,
12931-12940 |
21. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
22. | Towbin, H., Stachelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract] |
23. |
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. L.,
and Randall, R. J.
(1951)
J. Biol. Chem.
193,
265-275 |
24. | Cochran, B. H., Zullo, J., Verma, I. M., and Stiles, C. D. (1984) Science 226, 1080-1082[Medline] [Order article via Infotrieve] |
25. |
Ma, Q.,
Renzelli, A. J.,
Baldwin, K. T.,
and Antonini, J. M.
(2000)
J. Biol. Chem.
275,
12676-12683 |
26. |
Scott, L. J.,
and Hubbard, A. L.
(1992)
J. Biol. Chem.
267,
6099-6106 |
27. | Tanabe, T., Pricer, W. E., and Ashwell, G. (1979) J. Biol. Chem. 254, 1038-1043[Abstract] |
28. | Volk, B. A., Kreisel, W., Köttgen, E., Gerok, W., and Reutter, W. (1983) FEBS Lett. 163, 150-152[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Elovson, J.
(1980)
J. Biol. Chem.
255,
5816-5825 |
30. | Tauber, R., Kronenberger, C., and Reutter, W. (1989) Biol. Chem. Hoppe-Seyler 370, 1221-1228[Medline] [Order article via Infotrieve] |
31. | Mortimore, G. E. (1987) in Lysosomes: Their Role in Protein Breakdown (Glaumann, H. , and Ballard, F. J., eds) , pp. 415-444, Academic Press, Inc., New York |
32. |
Somsel,
Rodman, J.,
and Wandinger-Ness, A.
(2000)
J. Cell Sci.
113,
183-192 |
33. | Paulusma, C. C., Kothe, M. J., Bakker, C. T., Bosma, P. J., van Bokhoven, I., van Marle, J., Bolder, U., Tytgat, G. N., Oude, and Elferink, R. P. (2000) Hepatology 31, 684-693[Medline] [Order article via Infotrieve] |
34. | Rost, D., Karterbeck, J., and Keppler, D. (1999) Hepatology 29, 814-821[Medline] [Order article via Infotrieve] |
35. | Dombrowski, F., Kubitz, R., Chittattu, A., Wettstein, M., Saha, N., and Häussinger, D. (2000) Biochem. J. 348, 183-188[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Ameen, N. A.,
Martensson, B.,
Bourguinon, L.,
Marino, C.,
Isenberg, J.,
and McLaughlin, G. E.
(1999)
J. Cell Sci.
112,
887-894 |
37. |
Mukhopadhayay, S.,
Ananthanarayanan, M.,
Stieger, B.,
Meier, P. J.,
Suchy, F. J.,
and Anwer, M. S.
(1997)
Am. J. Physiol.
273,
G842-G848 |
38. |
Fushimi, K.,
Sasaki, S.,
and Marumo, F.
(1997)
J. Biol. Chem.
272,
14800-14804 |
39. |
Yao, X.,
Karim, S. M.,
Ramilo, M.,
Rong, Q.,
Thibodeau, A.,
and Forte, J. G.
(1996)
Am. J. Physiol.
271,
C61-C73 |
40. |
Pessin, J. E.,
Thurmond, D. C.,
Elmendorf, J. S.,
Coker, K. J.,
and Okada, S.
(1999)
J. Biol. Chem.
274,
2593-2596 |
41. | Ihrke, G., Neufeld, E. B., Meads, T., Shanks, M. R., Cassio, D., Laurent, M., Schroer, T. A., Pagano, R. E., and Hubbard, A. L. (1993) J. Cell Biol. 123, 1761-1775[Abstract] |
42. |
Shanks, M. R.,
Cassio, D.,
Lecoq, O.,
and Hubbard, A. L.
(1994)
J. Cell Sci.
107,
813-825 |
43. | Wakabayashi, Y., and Arias, I. M. (2000) Hepatology 32, 435 (abstr.) |