0090-9556/03/3104-482–490$7.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics
DMD 31:482–490, 2003
Vol. 31, No. 4
919/1052907
Printed in U.S.A.
MULTIPLE ALTERATIONS OF CANALICULAR MEMBRANE TRANSPORT ACTIVITIES IN
RATS WITH CCL4-INDUCED HEPATIC INJURY
IM-SOOK SONG, YOUNG-MI LEE, SUK-JAE CHUNG,
AND
CHANG-KOO SHIM
Department of Pharmaceutics, College of Pharmacy, Seoul National University, Seoul, Korea
(Received August 27, 2002; accepted January 6, 2003)
This article is available online at http://dmd.aspetjournals.org
ABSTRACT:
and Mrp2, respectively, was determined following an i.v. infusion to
rats. The uptake of each substrate into cLPM vesicles in the presence of ATP was also measured by a rapid filtration technique. As
the result of the CCl4-EHI, the protein level of transporters was
altered as a function of time in multiple manners; it was increased
by 3.6-fold for P-gp, unchanged for Bsep, and decreased by 73%
for Mrp2 at 24 h. The in vivo CLexc and the intrinsic uptake clearance into cLPM vesicles (CLint) at 24 h after the CCl4 injection
(CCl4-EHI24 h) were also influenced by the EHI in a similar manner;
they were increased by 1.8- and 1.9-fold for daunomycin, unchanged for taurocholate, and decreased by 41 and 39% for
E217␤G, respectively, consistent with multiple alterations in the
expression of the relevant transporters.
Primary active transporters in the liver canalicular membrane,
which contain the ATP binding cassette (ABC1), play an important
role as efflux pumps in the excretion of endogenous bile constituents
or xenobiotics into the bile canaliculi (Hooiveld et al., 2001). As
might be expected, certain types of liver diseases have an influence on
this hepatobiliary excretion. Experimental hepatic injury (EHI) induced by a single administration of carbon tetrachloride (CCl4) has
been widely used as a pathological model for liver diseases, since it is
known that CCl4 produces acute hepatocellular injury with centrilobular necrosis and steatosis (Recknagel, 1967). The effects of CCl4-EHI
on biochemical characteristics such as increased lipid peroxidation
(Recknagel, 1967) and the activities (Mourelle et al., 1987; Romero et
al., 1994) of glutamic oxaloacetic transaminase (GOT) and glutamic
pyruvic transaminase (GPT) and hepatobiliary excretion of xenobiotics (for example, sulfobromophthalein and leukotriene; Iga et al.,
1977; Wettstern et al., 1990) have been widely studied. Our earlier
study revealed that transport systems for some organic cations on the
sinusoidal membrane are prone to damage as the result of CCl4-EHI,
as demonstrated by a decrease in the in vitro maximal rates of hepatic
uptake and efflux without any influence on in vivo canalicular excretion clearance (Hong et al., 2000). In addition, a dose-dependent
inhibition of Na⫹/taurocholate cotransport (Ntcp) into sinusoidal
membrane vesicles by the presence of trichloroethylene and 1,1,2trichloro-1,2,2-trifluoroethane (all CCl4 analogs) has been reported
(Neghab et al., 1996). Recently, Geier et al. (2002) reported that
hepatobiliary organic anion transporters on the sinusoidal membrane
were regulated differently in acute toxic liver injury induced by CCl4;
mRNA levels were significantly decreased for Ntcp, Oatp1 (organic
anion transporting polypeptide1), and Oatp2, whereas the level remained unchanged for Oatp4.
Contrary to the cases of transport systems on the sinusoidal membranes, considerably less information is available on the effects of
CCl4-EHI on the canalicular transport system, which is believed to be
a key step in the vectorial transport of various xenobiotics from the
portal blood to the bile (Bossard et al., 1993; Han et al., 1999). Thus,
the objective of this study was to examine the effect of CCl4-EHI on
the expression and functional activity of representative ABC transporters on the canalicular membrane, including P-glycoprotein (P-gp),
a bile salt export pump (Bsep), and a multidrug resistance associated
protein2 (Mrp2). Western blot analysis was performed to evaluate the
expression of the transporters, and in vivo canalicular excretion and in
This work was supported, in part, by a Grant (02-PJ2-PG1-CH12–002) from
The Ministry of Health and Welfare, Republic of Korea.
1
Abbreviations used are: ABC, ATP binding cassette; EHI, experimental hepatic injury; CCl4, carbon tetrachloride; sGOT, serum glutamic oxaloacetic
transaminase; sGPT, serum glutamic pyruvic transaminase; Ntcp, Na⫹/taurocholate cotransport; P-gp, P-glycoprotein; Bsep, bile salt export pump; Mrp2,
multidrug resistance associated protein2; cLPM, canalicular liver plasma membrane; E217␤G, 17␤-estradiol-17␤-D-glucuronide; ALP, alkaline phosphatase;
MSB, membrane suspension buffer; PBST, phosphate-buffered saline solution
(pH 7.4) containing 0.1 (w/v) % Tween 20; HPLC, high performance liquid chromatography; TLC, thin-layer chromatography; LSC, liquid scintillation counting;
CLexc, in vivo canalicular excretion clearance; CLint, intrinsic clearance across the
canalicular membrane.
Address correspondence to: Dr. Chang-Koo Shim, Department of Pharmaceutics, College of Pharmacy, Seoul National University, San 56–1, Shinlim-dong,
Kwanak-gu, Seoul 151-742, Korea. E-mail: [email protected]
482
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The influence of CCl4-induced experimental hepatic injury (CCl4EHI) on the expression and transport activities of primary active
transporters on the canalicular membrane, including P-glycoprotein (P-gp), a bile salt export pump (Bsep) and a multidrug resistance associated protein2 (Mrp2), was assessed. CCl4-EHI was
induced by an intraperitoneal injection of CCl4 to rats at a dose of
1 ml/kg 24 h prior to the preparation of canalicular liver plasma
membrane (cLPM) vesicles and pharmacokinetic studies. The expression of each transporter was measured for the vesicles via
Western blot analysis at 6, 12, 24, 36, and 48 h after the injection of
CCl4. The in vivo canalicular excretion clearance (CLexc) of
[3H]daunomycin, [3H]taurocholate and [3H]17␤-estradiol-17␤-Dglucuronide (E217␤G), representative substrates of P-gp, Bsep,
EFFECT OF CCl4 ON CANALICULAR ABC TRANSPORTERS
vitro uptake into canalicular liver plasma membrane (cLPM) vesicles
for daunomycin, taurocholate, and 17␤-estradiol-17␤-D-glucuronide
(E217␤G) were measured to estimate the functional activity of the
relevant transporters. These compounds were selected because they
represent substrates for P-gp (Kamimoto et al., 1989; Hooiveld et al.,
2001), Bsep (Gerloff et al., 1998; Hooiveld et al., 2001), and Mrp2
(Morikawa et al., 2000; Hooiveld et al., 2001), respectively, and are
extensively excreted into bile via relevant transporters in rats [i.e.,
approximately 36% of an i.v. dose (10 ␮mole/kg) for daunomycin
(Catapano et al., 1988), 87% of an i.v. dose (54 ␮mole/kg) for
taurocholate (Ring et al., 1994), and 77% of an i.v. dose (180 pmol/
kg) for E217␤G (Morikawa et al., 2000)].
Materials and Methods
peroxidase-conjugated goat anti-mouse IgG for C219 and M2 III-6 (dilution
1:1000; Zymed Laboratories, South San Francisco, CA), and horseradish
peroxidase-conjugated goat anti-rabbit IgG for Antispgp (dilution 1:1000;
Amersham)] for 1 h at room temperature. After washing three times with 60 ml
of PBST, it was possible to visualize all proteins recognized by the antibodies
using an enhanced chemiluminescence detection system. Band intensities were
analyzed by densitometry using a software program, Quantity One (version
4.1; Bio-Rad, Hercules, CA).
In Vivo Biliary Excretion across Canalicular Membranes. After light
anesthesia with ketamine (50 mg/kg as i.p. dose; Yuhan Pharm. Co., Kyounggido, Korea), the femoral arteries and veins of rats were cannulated with PE-50
polyethylene tube and bile ducts with PE-10 polyethylene tube. Normal rats
and CCl4-pretreated (for 24 h) rats (i.e., CCl4-EHI24 h rats) were used throughout the in vivo excretion experiments. For the estimation of canalicular
excretion clearance (CLexc) of daunomycin, [3H]taurocholate and
[3H]E217␤G, rats received i.v. injection at bolus doses of 10 ␮mole/kg, 400
pmol/kg, and 160 pmol/kg, followed by i.v. infusion at rates of 10 ␮mole/h/kg,
400 pmol/h/kg (12 ␮Ci), and 160 pmol/h/kg (48 ␮Ci), for respective compounds. Blood samples (0.3 ml) were taken from the femoral artery at 30-min
intervals over a 3-h period, and bile was collected for 30-min period up to 3 h.
Plasma samples were separated from the blood samples by centrifuging at
3,000 rpm for 10 min. At 3 h (i.e., at the steady state) after each infusion start,
rats were sacrificed and the liver was isolated immediately. A 20% liver
homogenate was then prepared using normal saline, centrifuged at 3,000 rpm
for 10 min, and aliquots (100 ␮l) of the supernatant were collected for the
determination of the respective compounds. The in vivo CLexc of a compound
was calculated by dividing the biliary excretion rate by the liver substrate
concentration at the steady state.
Assay of Taurocholate, Daunomycin, and E217␤G in In Vivo Study. The
concentrations of taurocholate in plasma, liver (homogenates), and bile samples were quantified by liquid scintillation counting (LSC, Wallac 1409;
PerkinElmer Life Science Inc.). The concentration of daunomycin, which is
known to be metabolized in the body to daunomycinol (Pea et al., 2000), was
determined by high performance liquid chromatography (HPLC). A 100-␮l
aliquot of the biological samples (i.e., plasma, bile, and supernatant of the liver
homogenate) was deproteinized by the addition of MeOH (250 ␮l) containing
Adriamycin (internal standard, 1 ␮M). Ethyl acetate (1 ml) was then added, the
suspension was vigorously mixed for 5 min and then centrifuged at 10,000 rpm
for 5 min. An aliquot (1.2 ml) of the supernatant was transferred and evaporated using a Spinvac (Hanil Science, Seoul, Korea), and the residue was
reconstituted with the mobile phase (150 ␮l) used for HPLC (acetonitrile: 0.01
M phosphoric acid ⫽ 3: 7 v/v, pH 3.0). A 50-␮l aliquot of the reconstituted
solution was injected into the HPLC system, which consisted of a Hitachi
L-7110 pump (Hitachi, Japan), a Shimadzu RF 535 fluorescence detector
(Shimadzu, Japan), a Hitachi L-7200 autosampler, a Hitachi D-7500 integrator,
and a C18 column (Shisheido Capcell pak, 4.6 ⫻ 250 mm, 5-␮m particle size).
The flow rate of the mobile phase was set at 1 ml/min. The chromatogram was
monitored by fluorescence detection at an excitation wavelength of 470 nm
and an emission wavelength of 565 nm. The eluent resulted in sharp and well
resolved peaks corresponding to daunomycin, Adriamycin (internal standard),
and possible metabolites. The retention times of Adriamycin and daunomycin
were 4.9 and 8.3 min, respectively, under the conditions used. Calibration
curves of daunomycin were linear over the concentration range of 0.1– 4 ␮M
for plasma, bile, and liver samples, with respective correlation coefficients of
over 0.999.
The concentration of E217␤G, which is known to be metabolized mainly to
17␤-estradiol-3-sulfate-17-glucuronide (Meyers et al., 1980; Morikawa et al.,
2000) in the body, was determined by thin-layer chromatography (TLC)
followed by LSC. Plasma (100 ␮l) and supernatants of the liver homogenates
(100 ␮l) were deproteinized with methanol (250 ␮l), centrifuged at 10,000 rpm
for 5 min, and 300-␮l aliquots were evaporated using a Spinvac. The residues
were dissolved in methanol (20 ␮l), and 5-␮l aliquots were spotted onto silica
gel coated TLC plates (10 ⫻ 20 cm; Aldrich Chemical Co., Milwaukee, WI).
Bile specimens (5 ␮l) were directly spotted onto the TLC plates. TLC plates
were developed with chloroform/methanol/acetic acid (7:2:1 v/v) as the irrigant. Rf values of E217␤G and its major metabolites, 17␤-estradiol-3-sulfate17-glucuronide, were 0.45 and 0.1, respectively. The recovery of E217␤G from
plasma, liver, and bile samples in the TLC was over 95% for the concentration
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Materials. [ H]Taurocholate (2 Ci/mmol), [3H]17␤-estradiol-17␤-D-glucuronide (E217␤G, 44 Ci/mmol) and [3H]daunomycin (4.4 Ci/mmol) were
purchased from PerkinElmer Life Science Inc. (Boston, MA). C219 and M2
III-6, monoclonal antibodies to P-gp and Mrp2, respectively, were purchased
from Alexis Biochemical Co. (San Diego, CA), and Antispgp, a polyclonal
antibody to Bsep, was purchased from Kamiya Biomedical Co. (Seattle, WA).
An enhanced chemiluminescence detection system was purchased from Amersham Biosciences Inc. (Piscataway, NJ). Sucrose was purchased from Junsei
Chemical Co. (Tokyo, Japan). All other chemicals including reagents for
Western blot analysis and the vesicle uptake study were purchased from
Sigma-Aldrich (St. Louis, MO).
Induction of Experimental Hepatic Injury by CCl4. Male Sprague Dawley rats (250 –300 g; Dae-Han Biolink, Taejon, Korea) were injected intraperitoneally with a single dose of CCl4 (1 ml/kg) as a 50% (v/v) solution in olive
oil and then fasted for 6, 12, 24, 36, or 48 h. Water was fed ad libitum. Control
animals received a corresponding dose of olive oil using the same experimental
protocol. The activities of sGPT and sGOT were measured (Reitman and
Frankerl, 1957) using a commercial colorimetric determination kit (Yeoung
Dong Pharm. Co., Seoul, Korea). Experimental protocols involving animal
studies were reviewed by the Animal Care and Use Committee in College of
Pharmacy, Seoul National University according to the National Institutes of
Health guidelines (National Institutes of Health publication number 86 –23,
revised 1985) of “Guide for the Care and Use of Laboratory Animals”.
Preparation of cLPM Vesicles. cLPM vesicles were prepared from four
normal and CCl4-EHI rats according to the method of Inoue et al. (1983) as
described previously (Song et al., 1999). The purity of the vesicle preparations
was routinely assessed by measuring the relative enrichment of the activity of
alkaline phosphatase (ALP, Pekarthy et al., 1972), a marker enzyme for the
canalicular membrane, compared with that of crude homogenates. The protein
concentration of the vesicle preparation was measured by the Lowry method
(Lowry et al., 1951) using bovine serum albumin as a standard. To estimate the
relative proportion of the inside-out vesicles, the concentrations of the exposed
sialic acid of the vesicles were determined (Warren, 1959). Immediately after
their preparation, the cLPM vesicles were suspended in a membrane suspension buffer (MSB), which contained 250 mM sucrose, 10 mM Hepes, 10 mM
Tris, 10 mM MgCl2, and 0.2 mM CaCl2 (pH 7.4), to yield a protein concentration of 8 to 10 mg/ml. The suspension was stored at ⫺70 cC, for up to 2
weeks, until the uptake studies and Western blot analysis were carried out.
Western Blot Analysis. The vesicle preparations were diluted in a buffer
consisting of 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, and 50 mM Tris-HCl (pH 6.8) to yield a total protein concentration
of 1.25 ␮g/␮l. A 16-␮l aliquot of the suspension (equivalent to 20 ␮g of total
protein) and 10 ␮l of a solution of molecular weight markers (High Mw-SDS
calibration kit, Amersham Biosciences Inc.) were subjected to electrophoresis
on a 6% polyacrylamide gel with 0.1% SDS, and electrotransferred to a
nitrocellulose membrane (0.45 ␮m pore size; Amersham Biosciences Inc.).
The membrane was blocked with a phosphate-buffered saline solution (pH 7.4)
containing 0.1 (w/v) % Tween 20 (PBST) and 5% (w/v) nonfat dry milk (Seoul
Milk Co., Seoul, Korea) for 1 h at 37°C, and was probed for 18 h at 4°C with
the primary antibodies (dilution 1: 1000 each) C219 (Lee et al., 1996), M2 III-6
(Kool et al., 1997), and Antispgp (Lecureur et al., 2000) that recognize P-gp,
Mrp2, and Bsep, respectively. After washing three times with 60 ml of PBST,
the membrane was incubated with secondary antibodies [i.e., horseradish
3
483
484
SONG ET AL.
V0 ⫽
V max ⫻ S
Km ⫹ S
Where Vo is the initial uptake rate of the substrates (expressed as picomoles
per milligrams of protein per 30 seconds), S is the initial concentration of
substrates in the medium (␮M). Vmax and Km represent the maximal uptake rate
TABLE 1
Pathophysiological changes in rats 24 h after CCl4 (1 ml/kg) treatment
Weight
Body (g)
Liver (g)
Serum parameters
sGOT (unit/ml serum)
sGPT (unit/ml serum)
Liver homogenates
Protein (mg/g liver)
ALP (mmol/mg protein/h)
a
Normal
CCl4-EHI24 h
258 ⫾ 7.2
10.6 ⫾ 0.95
247 ⫾ 9.4
12.2 ⫾ 0.57*
86.8 ⫾ 10
84.0 ⫾ 6.6
997 ⫾ 22*
1184 ⫾ 210*
180 ⫾ 23
0.044 ⫾ 0.01
146 ⫾ 11*
0.056 ⫾ 0.01
a
All data represent the means ⫾ S.D. (n ⫽ 14).
* Statistically different from normal rats (P ⬍ 0.01).
and the medium concentration at half of maximal uptake rate, respectively. The
intrinsic clearance for the uptake (CLint) was obtained from Vmax/Km.
Data Analysis. All data are expressed in the form of the mean ⫾ S.D. A
two-way analysis of variance was performed to test differences in the temporal
uptake of each substrate between the transport conditions and treatments (i.e.,
normal and CCl4-EHI). The student’s t test was used to test differences in the
mean kinetic parameters (i.e., for in vitro and in vivo experiments) between the
treatments. In all cases, P ⬍ 0.01 was accepted as denoting a statistical
difference.
Results
Pathophysiological and Biochemical Changes by CCl4-EHI.
The effects of CCl4-EHI24 h (i.e., EHI at 24 h after the CCl4 administration) on various pathophysiological parameters are summarized in
Table 1. While the body weights were similar in both rats, the liver
weight was increased in CCl4-EHI24 h rats by 15%. EHI was confirmed by 11 and 14-fold increases in the activities of sGOT and
sGPT, respectively. The yield of protein from liver homogenates was
decreased by 19% as a result of the CCl4-EHI24 h, which is consistent
with the previously reported decrease in protein synthesis (Romero et
al., 1994). The ALP activity of the homogenate was unaffected by the
CCl4-EHI24 h.
Basic parameters for cLPM vesicles are summarized in Table 2.
The protein yield and ALP activity of cLPM vesicles were increased
by a factor of 43 and 54%, respectively, by the CCl4-EHI24 h, consistent with previously reported data (Mourelle et al., 1987). The ALP
activities in the vesicles from normal and CCl4-EHI24 h rats were
enriched by 52- and 63-fold, respectively, compared with the corresponding liver homogenates (compare Tables 1 and 2). In spite of the
differences in protein levels and ALP activity, the uptake of mannitol
by cLPM vesicles was about the same for both the normal and
CCl4-EHI24 h rats (Table 2), indicating a similar and negligible leakage of vesicle membranes. The proportion of inside-out configuration
of the vesicles was not affected by the CCl4-EHI24 h, consistent with
our previous reports (Song et al., 1999).
Western Blot Analysis. The expression of ABC transporters that
contain ATP binding cassettes (ABC) on canalicular membranes was
measured for cLPM vesicles by a Western blot analysis (Fig. 1).
Representative immunoblots for P-gp in cLPM vesicles, prepared
from livers of normal and CCl4-EHI rats, are shown in Fig. 1A. Bands
of approximately 170 kDa, the molecular size of P-gp (Kamimoto et
al., 1989), was observed for all the vesicle samples. The intensity of
the band varied with time (6, 12, 24, 36, and 48 h) after the CCl4
administration. Densitometric analysis (Fig. 1, A and A⬘) shows a
significant decrease in P-gp expression at 6 h after CCl4 administration, compared with cLPM vesicles from the normal liver (i.e., control). The expression levels at 12 h, however, exhibited a marked
increase (i.e., 2.1-fold of the control value). A maximal expression
(i.e., 3.6-fold increase) was observed at 24 h after the CCl4 adminis-
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range examined (0.1– 40 nM) regardless of the nature of the biological samples
(i.e., plasma, bile, and the supernatant of liver homogenate). Zones on the TLC
plates that correspond to an Rf of E217␤G were collected (by scraping) and the
radioactivity determined by LSC. The radioactivity associated with E217␤G
was 85 to 90, 45 to 50, and 65 to 70% of total radioactivity loaded on the TLC
plates for plasma, liver and bile samples, respectively, and the remaining
radioactivity could be attributed to a major metabolite of E217␤G, probably
17␤-estradiol-3-sulfate-17-glucuronide. No other substances exhibiting significant radioactivity were detected on the TLC plates.
In Vitro Vesicle Uptake Studies. The uptake of [3H]daunomycin, [3H]taurocholate, and [3H]E217␤G into cLPM vesicles was measured by a rapid
filtration technique (Song et al., 1999) using vesicles prepared from normal
and CCl4-EHI24 h rats. The uptake of [3H]daunomycin was measured as
follows. A frozen vesicle suspension was quickly thawed by immersion in a 37
°C water bath, re-vesicularized by passing it through a 25 gauge needle
20 times, and appropriately diluted with MSB to give 3 to 4 mg/ml of protein.
Ten microliters of the diluted suspension was preincubated in a test tube at 37
c
C for 4 min, and 40 ␮l of MSB, which contained 0.2 ␮M daunomycin (0.035
␮Ci) with or without the ATP-regenerating system (1.2 mM ATP, 3 mM
phosphocreatine, and 3.6 ␮g/100 ␮l creatine phosphokinase), was then added
to the diluted vesicle suspension. At predetermined times, the uptake was
quenched by the addition of 1 ml of an ice-cold solution of MSB containing 20
␮M daunomycin. The entire sample was then rapidly filtered through a
MF-MEMB filter (0.45-␮m pore size, 25-mm diameter; Seoul Science, Seoul,
Korea), which had been presoaked for 2 h in ice-cold MSB. The tube was
rinsed again with 1 ml of ice-cold MSB and then filtered. After washing with
10 ml of ice-cold MSB, the filter was dissolved in 4 ml of scintillation cocktail
(Ultimagold; PerkinElmer Life Science Inc.), and the radioactivity of the
mixture was determined by LSC. Presoaking and rinsing the filter with the
ice-cold MSB, which contains 20 ␮M daunomycin, resulted in a very small
level of nonspecific binding of daunomycin to the filter (i.e., negligible
radioactivity in the filter, data not shown). The amount of daunomycin in the
vesicles (expressed as picomoles per milligrams of protein) was plotted against
time. The ATP-dependent fraction that was taken up was estimated by the
difference in uptake between the two conditions (i.e., in the presence and
absence of ATP). The initial rate of uptake of daunomycin (i.e., ATP-dependent or -independent) was obtained from the linear portion (generally up to 1
min) of the temporal uptake profile. The concentration dependence of the
ATP-dependent initial uptake rate of daunomycin was examined for the
concentration range of 5 to 500 ␮M.
To determine the uptake into cLPM vesicles of taurocholate and E217␤G,
40 ␮l of the incubation medium containing 1 ␮M [3H]taurocholate (0.15 ␮Ci)
or 1 ␮M [3H]E217␤G (0.2 ␮Ci), with or without the ATP-regenerating system,
was added to the diluted vesicle suspension (10 ␮l). The concentration of cold
taurocholate or E217␤G in the relevant ice-cold MSB was 1 or 0.1 mM,
respectively. The concentration dependence of the ATP-dependent initial uptake rate of each substrate was examined for the concentration ranges of 5 to
500 ␮M for taurocholate and 0.5 to 200 ␮M for E217␤G.
To examine the issue of whether CCl4 influences the uptake of substrates
via direct interaction with transport systems, cLPM vesicles (2 - 2.5 mg/ml of
protein) from normal rats were incubated with MSB containing 1.5 mM CCl4
(Romero et al., 1994) at 37°C for 15 min, and the temporal uptake of 0.2 ␮M
[3H]daunomycin, 1 ␮M [3H]taurocholate and 1 ␮M [3H] E217␤G into the
vesicles, in the presence or absence of the ATP, was then determined as
described above.
To examine the concentration dependence, ATP-dependent initial uptake
rates of the substrates were plotted against the initial concentration of the
substrates in the medium, and the resulting profiles were fitted to MichaelisMenten equation (eq. 1, using a nonlinear regression analysis [Gauss-Newton
(Levenberg and Hartley), WinNonlin, version 3.1; Pharsight Co., Moutainview, CA].
EFFECT OF CCl4 ON CANALICULAR ABC TRANSPORTERS
TABLE 2
Changes in the basic parameters of cLPM vesicles at 24 h after CCl4
administration (1 ml/kg) to normal rats a
Protein (mg/g liver)
ALP (mmol/mg prot./h)
Mannitol uptake (% of medium)
Inside-out configuration (%)
Normal
CCl4-EHI24 h
0.140 ⫾ 0.05
2.28 ⫾ 0.94
3.85 ⫾ 0.98
30.2 ⫾ 8.6
0.200 ⫾ 0.03*
3.52 ⫾ 0.91*
3.28 ⫾ 0.41
29.9 ⫾ 9.1
a
All data represent the means ⫾ S.D. (n ⫽ 6).
* Statistically different from normal cLPM vesicles (P ⬍ 0.01).
cLPM vesicles were prepared from pooled liver homogenates from normal (n ⫽
4) and CCl4-EHI rats (n ⫽ 4) at 6, 12, 24, 36, and 48 h after an intraperitoneal
injection of CCl4 (1 ml/kg). Three different batches of vesicles were prepared using
different liver homogenate pools (i.e., the number of rats at each time point ⫽ 12,
the total number of rats used ⫽ 72). Lanes were loaded with each membrane vesicle
preparation (20 ␮g of total protein). The right lane was loaded with a high molecular
weight size marker. The target protein was detected using antibodies such as C219,
M2III-6, and Antispgp that recognize P-gp, Mrp2, and Bsep, respectively. Each data
point in the right column is expressed as the mean ⫾ S.D. for three different batches
of cLPM vesicle preparations.
tration. In 36 h, the expression decreased to levels similar to those of
the control. Representative immunoblots for Bsep in cLPM vesicles
are shown in Fig. 1B. Bands of approximately 150 kDa, the molecular
size of Bsep (Gerloff et al., 1998), were observed for all the vesicle
samples. Contrary to the case for P-gp, no change in the expression of
Bsep was observed by the CCl4 treatment up to 48 h (Fig. 1, B and
B⬘). The strongest band of approximately 190 kDa (Mottino et al.,
2000) was observed for Mrp2 (Fig. 1C). The density of the band
decreased as a function of time after CCl4 administration, exhibiting
a maximal decrease to 27% of control at 24 h and partial recovery to
60% in 48 h (Fig. 1, C and C⬘).
In Vivo Canalicular Excretion Clearance. To address the issue of
whether the altered expression of ABC transporters (Fig. 1) is reflected on the in vivo functional activity of the transporters, the effect
of CCl4-EHI on the canalicular excretion of representative substrates
of the transporters was examined. Since the rats exhibited maximal
change in the expression of P-gp and Mrp2 at 24 h after the CCl4
pretreatment (Fig. 1), subsequent in vivo experiments were performed
using rats at 24 h after the pretreatment. An i.v. infusion of taurocholate and E217␤G was performed at tracer doses to avoid their
possible interactions with endogenous taurocholate and E217␤G
(Morikawa et al., 2000) in canalicular excretion. As the result, the
plasma concentration of these compounds remained in the nanomolar
range (Table 3). The well known cholestatic effect of E217␤G (Meyers et al., 1980; Morikawa et al., 2000) was not apparent at this plasma
level, demonstrated by comparable bile flow rates among normal rats
[i.e., 65.5 ⫾ 11 ␮l/min/kg (mean ⫾ S.D., n ⫽ 6) for daunomycin,
55.5 ⫾ 9.5 ␮l/min/kg (mean ⫾ S.D., n ⫽ 6) for taurocholate, 52.2 ⫾
14 ␮l/min/kg (mean ⫾ S.D., n ⫽ 6) for E217␤G, and 58.4 ⫾ 6.7
␮l/min/kg (mean ⫾ S.D., n ⫽ 3) for control]. Moreover, bile flow was
not influenced by the CCl4-EHI24 h regardless of the compounds
infused (data not shown).
A steady-state plasma concentration and biliary excretion of daunomycin, taurocholate and E217␤G were attained within 60 min after the
initiation of constant rate i.v. infusion and a simultaneous i.v. bolus
injection of the compounds in both normal and CCl4-EHI24 h rats (Fig.
2). Thus, the steady-state value for each rat was read from the average
of the last four data points and is expressed in Table 3 as the mean ⫾
S.D. of six rats. No significant differences in the steady state plasma
concentration and biliary excretion rate of daunomycin, a representative substrate for P-gp (Kamimoto et al., 1989; Hooiveld et al., 2001),
were found between the normal and CCl4-EHI24 h rats. However, the
CLexc (i.e., in vivo canalicular excretion clearance calculated from
dividing biliary excretion rate by liver concentration at the steady
state) of daunomycin was increased by 1.6-fold, whereas the steadystate liver concentration of daunomycin was decreased by 42% by the
EHI (Table 3).
The steady-state plasma concentration of taurocholate, a representative substrate for Bsep (Gerloff et al., 1998; Hooiveld et al., 2001),
was increased by 1.3-fold, but no changes in liver concentration or the
biliary excretion rate were detected by the CCl4-EHI24 h, resulting in
a constant value for CLexc. On the other hand, the CCl4-EHI24 h
increased the plasma (1.7-fold) and liver concentrations (1.9-fold) of
E217␤G, a representative substrate for Mrp2 (Morikawa et al., 2000;
Hooiveld et al., 2001), without influencing biliary excretion rate of the
compound, thereby resulting in 41% decreases in the CLexc of the
compound (Table 3). In summary, changes in the in vivo CLexc of
these substrates were generally consistent with changes in the expression of relevant transporters CCl4-EHI24 h rats (Fig. 1), except for the
CLexc of daunomycin, which exhibited a less significant increase (i.e.,
1.8-fold) than expected from the 3.6-fold increase in the expression of
P-gp (Fig. 1).
Alterations in the ATP-dependent Uptake of Daunomycin, Taurocholate and E217␤G into cLPM Vesicles. Certain pathophysiological changes by EHI, such as decreased metabolic activity (Olatunde Farombi, 2000) and an increased plasma level of endogenous
compounds (e.g., corticosterone; Huang et al., 2001), might interfere
with the in vivo transport of relevant substrates. To exclude this
possibility, the uptake of these substrates into cLPM vesicles from
normal and CCl4-EHI24 h rats was examined. Figure 3A shows the
result for daunomycin. The uptake of daunomycin was significantly
increased by the presence of ATP. The uptake in the absence of ATP
was not influenced by the CCl4-EHI24 h, whereas the uptake in the
presence of ATP was increased significantly for cLPM vesicles from
CCl4-EHI24 h rats. Figure 3A⬘ shows the concentration (5–500 ␮M)
dependence of the ATP-dependent uptake rate of daunomycin. Consistent with Fig. 3A, a much higher rate of uptake was observed by the
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
FIG. 1. Representative immunoblots of P-gp (A), Bsep (B), and Mrp2 (C)
proteins, and the relative densities of immunostained bands of P-gp (A⬘), Bsep
(B⬘), and Mrp2 (C⬘) in cLPM vesicles from normal and CCl4-EHI rats.
485
486
0.263 ⫾ 0.011 nM*
0.792 ⫾ 0.19 nM*
1.60 ⫾ 0.13 pmol/min/kg
2.12 ⫾ 0.49*
All data represent the means ⫾ S.D. (n ⫽ 6).
CLexc was calculated from dividing the excretion rate by the liver substrate concentration at the steady state.
* Significantly different compared to normal rats (P ⬍ 0.01).
b
a
0.350 ⫾ 0.013 nM*
1.26 ⫾ 0.13 nM
5.44 ⫾ 1.2 pmol/min/kg
4.22 ⫾ 0.64
2.00 ⫾ 0.13 ␮M
21.1 ⫾ 4.0 ␮M*
7.99 ⫾ 1.5 nmol/min/kg
0.353 ⫾ 0.061*
Plasma concentration (P)
Liver concentration (L)
Biliary excretion rate
CLexc(ml/min/kg)b
2.30 ⫾ 0.24 ␮M
36.1 ⫾ 3.3 ␮M
7.14 ⫾ 0.79 nmol/min/kg
0.198 ⫾ 0.015
0.288 ⫾ 0.027 nM
1.39 ⫾ 0.30 nM
5.20 ⫾ 0.62 pmol/min/kg
3.74 ⫾ 0.91
0.151 ⫾ 0.012 nM
0.409 ⫾ 0.12 nM
1.35 ⫾ 0.15 pmol/min/kg
3.57 ⫾ 0.38
CCl4-EHI24 h
CCl4-EHI24 h
Taurocholate (400 pmol/h/kg)
Normal
CCl4-EHI24 h
Daunomycin (10 ␮mol/h/kg)
Normal
TABLE 3
FIG. 2. Plasma concentrations and biliary excretion rates of daunomycin (A and
A⬘, respectively), taurocholate (B, B⬘) and E217␤G (C, C⬘) in normal (E) and
CCl4-EHI24 h (F) rats following i.v. bolus dose and i.v. infusion.
Doses of daunomycin, taurocholate, and E217␤G were 10 ␮mole/kg and 10
␮mole/h/kg, 400 pmol/kg and 400 pmol/h/kg (12 ␮Ci), and 160 pmol/kg and 160
pmol/h/kg (48 ␮Ci) (for respective i.v. bolus doses and infusion rates), respectively.
Each point represents the mean ⫾ S.D. of six different rats.
CCl4-EHI24 h. A kinetic analysis revealed a 1.8-fold increase in the
value of Vmax for the uptake of daunomycin into cLPM vesicles from
the CCl4-EHI24 h rats, compared with those from normal rats, whereas
the values of Km for vesicles from both rats were comparable (Table
4). As a result, a 1.9-fold increase in the value of CLint was observed
by the CCl4-EHI24 h for daunomycin (Table 4), highly consistent with
the increase in CLexc in the in vivo study (i.e., a 1.8-fold increase).
The increase in the value of Vmax under a constant value of Km by the
CCl4-EHI24 h appears to be consistent with the increase in the expression of P-gp by the CCl4-EHI24 h (Fig. 1, A and A⬘), although it is less
prominent than expected from the 3.6-fold increase in the expression
of P-gp (Fig. 1)
Temporal profiles for the uptake of 1 ␮M taurocholate into cLPM
vesicles are shown in Fig. 3B. The uptake of taurocholate was greatly
increased by the presence of ATP. However, no differences in the
uptake of taurocholate were observed between normal and CCl4EHI24 h rats, regardless of the presence of ATP. The uptake of
taurocholate in the presence of ATP was determined for a taurocholate
concentration range of 5 to 500 ␮M. Consistent with Fig. 3B, no
apparent effect of CCl4-EHI24 h was observed in the concentrationuptake rate profiles (Fig. 3B⬘). As a result, similar kinetic parameters
(i.e., Km, Vmax, and CLint) were obtained for the vesicular uptake of
taurocholate for both normal and CCl4-EHI24 h rats (Table 4). The
similarity in Vmax values between normal and CCl4-EHI24 h rats is
consistent with the results of the Western blot analysis for Bsep (Fig.
1, B and B⬘).
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
Summary of the Effects of CCl4 on the Pharmacokinetic Parameters of daunomycin, taurocholate, and E217 ␤G at steady-state conditionsa.
Normal
E217 ␤G (160 pmol/h/kg)
SONG ET AL.
EFFECT OF CCl4 ON CANALICULAR ABC TRANSPORTERS
487
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FIG. 3. Effects of CCl4–EHI on the ATP-dependent uptake of 0.2 ␮M [3H]daunomycin (A), 1 ␮M [3H]taurocholate (B), and 1 ␮M [3H]E217␤G (C) into cLPM
vesicles from normal (E, ƒ) and CCl4–EHI rats (F, ) in the presence (E, F) or absence (ƒ, ) of ATP. Concentration dependence in the ATP-dependent uptake of
daunomycin (A⬘, 5–500 ␮M), taurocholate (B⬘, 5–500 ␮M) and E217␤G (C⬘, 0.5–200 ␮M).
ATP-dependent fraction of the uptake (A⬘, B⬘, and C⬘, F) was obtained by subtracting the uptake rate of a substrate in the absence of ATP from that in the presence
of ATP. Curved lines were generated using the estimated kinetic parameters in Table 4. Each data point is expressed as the mean ⫾ S.D. of triplicate measurements from
three different batches of membrane vesicle preparations. 多, statistically different from normal cLPM vesicles (P ⬍ 0.01).
Temporal profiles of the uptake of [3H]E217␤G into cLPM vesicles
from normal and CCl4-EHI24 h rats are shown in Fig. 3C. The uptake
was greatly increased by the presence of ATP. The uptake in the
absence of ATP was not influenced by the CCl4-EHI24 h, whereas the
ATP-dependent uptake was much greater than the ATP-independent
uptake and was decreased significantly by the CCl4-EHI24 h. A concentration dependence in the ATP-dependent uptake of [3H]E217␤G
was found for the concentration range of 0.5 to 200 ␮M (Fig. 3C⬘). A
kinetic analysis of the ATP-dependent uptake revealed a 39% decrease in the value of Vmax without affecting the value of Km, leading
to a 39% decrease in the value of CLint as a result of the CCl4-EHI24 h
(Table 4). This result appears to be consistent with the results of the
Western blot analysis for Mrp2, which exhibited a 73% decrease in the
expression (Fig. 1, C and C⬘).
In summary, the CCl4-EHI24 h increased the vesicular uptake of
daunomycin, did not influence the uptake of taurocholate, and decreased the uptake of E217␤G, in the presence of ATP, all highly
consistent with the effect of the CCl4-EHI24 h on the expression of the
responsible transporters (i.e., P-gp, Bsep and Mrp2, respectively, Fig.
1) and the in vivo canalicular excretion (i.e., CLexc, Table 3).
Effect of Direct Contact of CCl4 on the Uptake of Substrates
into cLPM Vesicles. To examine the issue of whether the above
488
SONG ET AL.
TABLE 4
Kinetic parameters for the ATP-dependent uptake of daunomycin, taurocholate,
and E217 ␤G by cLPM vesiclesa
Daunomycin
Taurocholate
E217 ␤G
Vmax (nmol/mg protein/30 s)
Km (␮M)
CLintb (␮l/mg protein/30 s)
Vmax (nmol/mg protein/30 s)
Km (␮M)
CLint (␮l/mg protein/30 s)
Vmax (nmol/mg protein/30 s)
Km (␮M)
CLint (␮l/mg protein/30 s)
Normal
CCl4-EHI24 h
3.69 ⫾ 0.36
49.8 ⫾ 15
77.4 ⫾ 15
0.950 ⫾ 0.79
75.9 ⫾ 4.2
12.5 ⫾ 0.49
0.166 ⫾ 0.017
4.22 ⫾ 0.88
40.6 ⫾ 9.2
6.72 ⫾ 0.66*
44.9 ⫾ 4.1
149 ⫾ 3.7*
1.02 ⫾ 0.45
80.4 ⫾ 3.8
12.7 ⫾ 0.07
0.102 ⫾ 0.016*
4.66 ⫾ 2.3
24.6 ⫾ 8.0*
All data represent the means ⫾ S.D. (n ⫽ 3).
CLint was calculated by dividing Vmax by Km.
* Statistically different from normal cLPM vesicles (P ⬍ 0.01)
a
b
FIG. 4. Effect of the direct addition of 1.5 mM CCl4 on the ATP-dependent
uptake of 0.2 ␮M [3H]daunomycin (A), 1 ␮M [3H]taurocholate (B), and 1 ␮M
[3H]E217␤G (C) in the absence (ƒ, ) and presence (E, F) of ATP.
Discussion
cLPM vesicles from normal rats were preincubated with (, F) or without CCl4
(ƒ, E) at 37°C for 15 min. Each point represents the mean ⫾ S.D. of triplicate
measurements from three different batches of membrane vesicle preparations. 多,
statistically different from control (P ⬍ 0.01).
Western blot analysis (Fig. 1) indicates that the expression level of
ABC transporters on the canalicular membrane, except for Bsep,
varies as a function of time after the administration of CCl4 to rats,
exhibiting maximal changes at 24 h (Fig. 1). Most interestingly, the
effect of CCl4-EHI24 h on the expression levels differed significantly
depending on the canalicular transporters; the expression level was
increased for P-gp, remained unchanged for Bsep, and was decreased
for Mrp2. These results for P-gp and Bsep are consistent with previous
reports (Huang et al., 2001; Geier et al., 2002), whereas the result for
Mrp2 is contrary to data reported by Geier et al. (2002), in which no
change in the expression level of Mrp2 was reported for the liver
microsomes of CCl4-EHI24 h rats. In the present study, Western blot
analysis of transporters was performed for cLPM vesicles instead of
liver microsomes (Geier et al., 2002). This difference (i.e., vesicles
and microsomes) might be involved in the discrepancy between the
two studies in the expression level of Mrp2.
In the present study, Antispgp (Lecureur et al., 2000), M2III-6
(Kool et al., 1997), and C219 (Lee et al., 1996) were used as antibodies for the immunostaining of Bsep, Mrp2, and P-gp, respectively.
Antispgp does not cross react with the mdr1a gene product (i.e., P-gp)
(Lecureur et al., 2000), and M2III-6 does not with gene products of
mdr1a and mrp2 isoforms (i.e., Mrp1, Mrp3, and Mrp5) (Kool et al.,
1997). C219, the most widely used antibody for P-gp (Lee et al.,
1996), on the other hand, exhibits a partial cross reactivity to other
proteins such as Mdr2 and Bsep (Van Den Elsen et al., 1999). The fact
that the level of Mdr2 appeared to be increased in 24 h after the CCl4
pretreatment (from the mRNA level of mdr2; Nakasukasa et al., 1993)
suggests that immunostaining using C219 might lead to an overestimation of P-gp levels. In other words, a 3.6-fold increase in the level
of P-gp by the CCl4-EHI24 h (Fig. 1) might be an overestimation, at
least in part.
The effect of the CCl4-EHI24 h (i.e., changes in the expression of the
ABC transporters on the canalicular excretion of representative substrates of the transporters was examined in vivo. The CLexc values for
the substrates were altered multiply (Table 3), consistent with the
changes in the expression level of relevant transporters (Fig. 1).
However, the change in the CLexc value for daunomycin (i.e., 1.9-fold
increase; Table 3) was not as significant compared with the changes
in the expression of P-gp (i.e., 3.6-fold increase; Fig. 1A⬘). This
discrepancy could be related to the cross reactivity of C219 (Van Den
Elsen et al., 1999) or to interference by certain endogenous factors in
CCl4-EHI24 h rats on canalicular excretion (Olatunde Farombi, 2000;
Huang et al., 2001).
To exclude possible interference by endogenous factors, the in vitro
uptake of relevant substrates into cLPM vesicles was examined.
Results from the in vitro uptake study were fairly consistent with the
expression of relevant transporters (Fig. 1) as demonstrated by Vmax
and Km values in Table 3. The CLint was 1.9-fold increased for
daunomycin, remained unchanged for taurocholate, and was 39%
decreased for E217␤G. An analysis of the uptake studies revealed that
the changes in the CLint are solely attributable to changes in the Vmax
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
observed effects of CCl4-EHI24 h are attributable to direct interactions
of the responsible transport systems with CCl4, the uptake of 0.2 ␮M
daunomycin, 1 ␮M taurocholate, and 1 ␮M E217␤G into cLPM
vesicles, following the incubation of normal cLPM vesicles with 1.5
mM CCl4 at 37°C for 15 min, was assessed in the presence and
absence of ATP. The uptake of daunomycin into normal vesicles was
decreased significantly by incubation in the presence of CCl4 and
ATP (Fig. 4A). This is contrary to the results obtained for the uptake
of daunomycin into cLPM vesicles that were prepared from CCl4EHI24 h rats (Fig. 3A), indicating that CCl4, in the body, affects the
ABC transporters on the canalicular membrane in an indirect manner.
On the other hand, the effect of direct contact of CCl4 on the uptake
of taurocholate and E217␤G into cLPM vesicles was similar to that of
CCl4-EHI24 h on the uptake of these substrates, regardless of the
presence of ATP (Fig. 3, B and C). Despite this similarity, however,
CCl4-EHI seems to influence the ABC transporters on the canalicular
membrane in a manner that is distinct from the direct contact of CCl4
with the membrane, based on the result of the uptake of daunomycin.
EFFECT OF CCl4 ON CANALICULAR ABC TRANSPORTERS
FIG. 5. Summary of the effect of the CCl4–EHI24 h on the expression of the
primary active transporters on the canalicular membrane and CLint and CLexc of
relevant substrates.
with the level of relevant mRNAs, since the level of mdr 1a and mdr
1b transcripts was reported to be continuously increased for up to 5
days after the CCl4 administration (Nakasukasa et al., 1993), whereas
the expression of the relevant transporter, P-gp, exhibited a maximal
expression at 24 h after the CCl4 administration in the present study
(Fig. 1, A and A⬘). Thus, post-transcriptional regulation appears to
influence, at least in part, the level of transporters on the canalicular
membrane. Similar discrepancies in the expression of transporters and
responsible mRNAs have been reported for Bsep and Mrp2 in ethynylestradiol-induced cholestasis rats (Trauner et al., 1997; Lee et al.,
2000).
One of the most important findings of the present study is the
multiple alterations in the expression and activities of canalicular
membrane transporters by the CCl4-EHI24 h (Fig. 5). A multiple effect
of CCl4 has also been demonstrated for the expression of mRNAs and
proteins of the sinusoidal transporters (i.e., Ntcp, Oatp1, Oatp2, and
Oatp4, Geier et al., 2002) as described in the Introduction. The
physiological meaning of this type of influence of CCl4-EHI, especially as host defense mechanisms of biological systems against
diseases and xenobiotics, should be carefully considered. Regardless
of the underlying mechanisms, the multiple (i.e., transporter and
membrane selective) effects of the CCl4-EHI should be taken into
account in understanding of hepatic injuries. Special care should be
taken concerning the increase in the expression and functional activity
of certain transporters (i.e., P-gp, in this study), because it might be
contrary to our general understanding that hepatic injuries may damage membrane transporters both in terms of expression and function.
In addition, it is noteworthy that the expression of ABC transporters
on the canalicular membrane may vary depending on the types of EHI,
as evidenced by the decrease in the level of both Bsep and Mrp2 by
ethynylestradiol-induced EHI (Bossard et al., 1993; Lee et al., 2000).
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The effect of CCl4-EHI24 h on the expression and functional activity
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In general, the effect of CCl4-EHI24 h on the ABC transporters, with
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functional activity of each transporter was in an identical direction for
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to represent the mechanism involved in the indirect effect of the CCl4
pretreatment.
Although the expression of transporters was changed by the CCl4EHI (Fig. 1), the expression does not appear to be necessarily parallel
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