1 Graduate Center for Toxicology, Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536 - 0305; and 2 Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, CH-8091 Zurich, Switzerland
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ABSTRACT |
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The expression of hepatic multidrug resistance-associated protein (Mrp)1, 2, 3, and 6 and organic anion transporting polypeptides (Oatp)1 and 2 were examined in control and 20- to 21-day pregnant rats. Western analysis showed that expression of Oatp2 was decreased 50% in pregnancy, whereas expression of Oatp1 did not change. Expression of Mrp2 protein determined by Western analysis of total liver homogenate decreased to 50% of control levels in pregnant rats, consistent with studies using plasma membranes. Confocal immunohistochemistry showed that Mrp2 expression was confined to the canalicular membrane in both control and pregnant rats and was not detectable in intracellular compartments. In isolated perfused liver, the biliary excretion of 2,4-dintrophenyl-glutathione was significantly decreased in pregnancy, consistent with decreased expression of Mrp2. The expression of the basolateral transporter Mrp1 was not altered in pregnancy, whereas expression of Mrp6 mRNA was decreased by 60%. Expression of Mrp3 was also decreased by 50% in pregnant rat liver, indicating differential regulation of Mrp isoforms in pregnancy. These data also demonstrate that decreased Mrp2 expression is not necessarily accompanied by increased Mrp3 expression.
organic anion transporting polypeptides
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INTRODUCTION |
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A KEY FUNCTION OF THE LIVER is the production of bile and biliary secretion of many endogenous and exogenous substances. These compounds are taken up across the sinusoidal membrane; biotransformed into glutathione, glucuronate, or sulfate conjugates in hepatocytes; and then excreted into the bile across the canalicular membrane. The hepatic uptake of bile salts is largely dependent on the Na+/taurocholate cotransporting polypeptide, whereas uptake of many other amphipathic organic anions is primarily dependent on the Na+-independent organic anion transporting polypeptides (Oatps) (13, 19, 35). Under physiological conditions, these conjugates are secreted across the canalicular membrane into bile by an ATP-dependent conjugate export pump, the canalicular organic anion transporter or multidrug resistance-associated protein 2 (MRP2/Mrp2; ABCC2) (11, 18). At least seven other members of the MRP/Mrp family have been identified, all of which confer multidrug resistance (3, 17, 23, 26). Mrp1, 2, 3 and 6 are normally expressed in both human and rodent livers, whereas their abundance and localization vary. Mrp1 and 3 are present in very low levels at the lateral membrane of hepatocytes under normal physiological conditions, whereas Mrp2 is abundant and functions as an important conjugate export pump on the canalicular membrane of hepatocytes (28, 36, 39, 45). Mrp6 was recently cloned in rat liver and characterized as a lateral and canalicular transporter (31). A physiological function for MRP/Mrp 4 and 5 has not yet been identified; however, both are able to transport organic anions and nucleotide analogs (4, 20, 43, 51).
Pregnancy is one of the major physiologically stressful events in which
the transport processes in the liver are dramatically altered. Previous
studies (6) have shown that Mrp2 protein expression is
significantly decreased in pregnancy, consistent with the decreased
biliary excretion of organic anions at this time. The ability of the
liver to concentrate the glucuronide conjugate of the hydroxylated
metabolite of phenytoin in bile is impaired in pregnancy, resulting in
its accumulation in blood (48). Under conditions where the
function of Mrp2 is hereditarily deficient in Eisai hyperbilirubinemic
or transporter mutant (TR) rats, or in chronic bile duct
ligation (CBDL)-induced cholestasis, the basolateral isoform Mrp3 is
dramatically induced (9, 15, 45). Mrp1 expression is
increased during hepatocyte proliferation and after LPS treatment,
which also inhibit Mrp2 expression (39, 50). These
basolateral isoforms of the Mrp family (Mrp1 and 3) are proposed to
mediate the secretion of conjugates from hepatocytes into blood,
particularly under pathophysiological conditions associated with
impairment of Mrp2 (25). Mrp2 substrates include
glutathione conjugates [e.g.,
2,4-dinitrophenyl-S-glutathione (DNP-SG) and leukotriene
C4 (LTC4)], glucuronide conjugates [e.g.,
estradiol 17-
-D-glucuronide (E217G)], and
sulfate conjugates of certain bile salts (e.g.,
taurolithocholate-3-sulfate), and are very similar to Mrp1 substrates
(21, 22, 24, 29, 46). Mrp3 also transports glucuronide
conjugates such as E217G as its substrates, whereas glutathione conjugates are poorly transported by this carrier (16).
Therefore, we examined expression of hepatic Mrp1 and Mrp3 in pregnant rats, compared with female controls, to determine whether their expression is altered under this physiological condition when Mrp2 is downregulated. The expression of Mrp6 and Oatp 1 and 2 was also examined. Finally, hepatic transport of DNP-SG was examined in the isolated perfused liver after infusion of the lipophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB).
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MATERIALS AND METHODS |
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Reagents. Proteinase inhibitors (PMSF, antipain, aprotinin, pepstatin A, and leupeptin) and CDNB were obtained from Sigma (St. Louis, MO). CDNB was recrystallized from ethanol and water (3:2 vol/vol) before use (12). All other chemicals were of analytical grade and were from Life Technologies (Rockville, MD), Fisher Scientific (Pittsburgh, PA), or Sigma. The GSH conjugated derivative of CDNB, DNP-SG, was synthesized using 1-fluoro-2,4-dinitrobenzene and GSH, as described (44).
Antibodies. Mouse monoclonal antibodies against the carboxyl terminus of human MRP2 (M2III-6) and rat monoclonal antibody against an internal epitope of human MRP1 (MRPr1) were purchased from Alexis Biochemicals (San Diego, CA). Because of the similar sequences of immunogen in rat and human, M2III-6 and MRPr1 also recognize rat Mrp2 and 1, respectively. The rabbit polyclonal antibody against rat Mrp3 was raised to a fusion protein containing amino acid 838-973 of the deduced rat Mrp3 amino acid sequence (36). Polyclonal rabbit anti-Zonula occludens 1 was purchased from Zymed Laboratory (South San Francisco, CA). Rabbit anti-serum against rat Oatp1 and 2 were prepared as described previously (10, 38).
Animals.
Female Sprague-Dawley rats (Harlan Industries, Indianapolis, IN) were
used throughout the study. The rats had free access to food and water
and were maintained on a 12:12-h automatically timed light/dark cycle.
Pregnant rats were timed according to the first day that sperm were
detected (day 0). Age-matched nonpregnant female rats were
used as controls and were compared with timed pregnant rats (20-21
days pregnant). TR rats (180-240 g), a gift from the
Academic Medical Center, Amsterdam, the Netherlands, were bred in our
animal facility. All procedures involving animals were conducted in
accordance with National Institutes of Health guidelines for the care
and use of laboratory animals and were approved by the Institutional
Animal Care and Use Committee of the University of Kentucky.
Single-pass perfused liver.
This experiment was designed to determine hepatic excretion of DNP-SG
from control and pregnant rats. Rats were anesthetized with 1 g/kg
urethane ip. The liver was perfused via the portal vein with
Krebs-Henseleit buffer consisting of (in mM): 118.5 NaCl, 24.9 NaHCO3, 1.2 KH2PO4, 1.19 MgSO4, 4.74 KCl, and 1.27 CaCl2, pH 7.4, at a
flow rate of 3.6-4.3
ml · min1 · g
1 liver in a
single-pass perfusion system as described previously (30).
The bile duct was cannulated with polyethylene-10 tubing. Perfusate was
oxygenated with 95% O2-5% CO2 and maintained
at 42°C so that the liver was maintained at 36 ± 1°C.
Taurocholate (10 µM) was infused throughout the experiment to
maintain stable bile flow. After an initial 10-min equilibration time,
CDNB (30 µM in 1:2,000 dimethylsulfoxide: Krebs-Henseleit buffer,
final concentration), the precursor of DNP-SG, was infused into the portal vein cannula for 30 min. The liver was then perfused with blank
Krebs-Henseleit buffer for an additional 40 min. Bile was collected
every 5 min throughout the experiment and the volume was determined
gravimetrically assuming a density of 1.0. Perfusate outflow was also
collected every 5 min. DNP-SG content was determined in bile and
perfusate outflow by HPLC. The HPLC system used was a Waters model
M-6000 (Waters, Milford, MA). Isocratic elution was performed
with a C18 column (µBondapak; Waters) with a mobile phase of
acetonitrile: 0.01% H3PO4 (1:3, vol/vol) at a
flow rate of 1.0 ml/min (14). DNP-SG was detected at 365 nm and was quantified by means of a standard curve.
Preparation of whole liver homogenate.
Livers were removed from control, 20-21 days pregnant, or
TR rats, rinsed with ice-cold PBS, cut into several
pieces, frozen in liquid nitrogen, and stored at
80°C. Frozen rat
liver pieces (
1 g) were homogenized in 4 vol of homogenization
buffer (10 mM Tris · HCl, pH 7.6, 140 mM NaCl, containing
proteinase inhibitors). Homogenate was mixed with an equal volume of
the above homogenization buffer containing 2% Triton X-100, and
rotated end-over-end at 4°C for 2 h. The homogenate was
centrifuged at 30,000 g for 30 min at 4°C, and the supernatant was
saved. The resulting pellet was suspended in 1 ml of homogenization
buffer containing 1% Triton X-100, rotated end-over-end at 4°C for
2 h, and centrifuged as described above. The above two
supernatants were combined, stored at
80°C, and used as whole liver
homogenate samples.
Immunoblot analysis.
Whole rat liver homogenate was suspended in sample loading buffer (52.5 mM Tris · HCl, pH 6.8, 2% SDS, 0.0025% bromophenol blue, 10%
glycerol, 2.5% -mercaptoethanol). Samples (10-20 µg; without
boiling) were separated by an 8 or 10% SDS-PAGE and transferred to a
nitrocellulose membrane. Membranes were blocked with Tris-buffered 5%
nonfat milk overnight at 4°C and then incubated with one of the
following antibodies: anti-Mrp2 (1:5,000 dilution), anti-Mrp3 (1:2,000
dilution), anti-Mrp1 (1:3,000) dilution, anti-Oatp2 (1:3,000 dilution),
and anti-Oatp1 (1:2,000 dilution) for 1-2 h at room temperature.
The blots were then washed three times in a TBS-Tween solution (10 mM
Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) and incubated with the
secondary antibodies (peroxidase-conjugated anti-rabbit or anti-mouse
antibodies) for 1-2 h. The blots were then washed again four times
in TBS-Tween solution. The labeled blots were visualized using the
enhanced chemiluminescence detection system (ECL Plus, Amersham
Pharmacia Biotech, Little Chalfont, Buchinghamshire, UK) and scanned
using a Molecular Dynamics phosphorimager for quantitation using
ImageQuant software.
Real-time PCR. Total RNA was prepared from rat livers according to Chomczynski and Sacchi (7). cDNA was produced from 4 µg of total RNA by using the SuperScript Preamplification System for first strand cDNA synthesis according to the manufacturer's instructions (Invitrogen, Carlsbad, CA) in a volume of 20 µl. Real-time quantitative PCR was performed on the cDNA sample using the Roche Molecular Biochemical's LightCycler System (Roche Diagnostics, Indianapolis, IN). The following primer pairs were used: Mrp1: 5'-CCTTTTCCTGTGCAATCATGTA-3' and 5'-AGAACCTCTGCACAAAGAAGTA-3'; Mrp3: 5'-GTGCTGAAGAATTTGACTCTG-3' and 5'-GACCAGGACCCGGTTGTAGTC-3'; Mrp6: 5'-CTGCTTCAGGAGAACACAGAT-3' and 5'-CTTGAAGTAGACAGCTTGGGCT-3'; resulting in amplified products of 461, 573, and 677 bp, respectively. Individual glass capillaries were filled with a solution containing 2 µl of cDNA template, 1.6 µl of MgCl2 (25 µM), 1.2 µl of specific primer pairs (10 µM each), 2 µl of SYBR Green Master solution, and 13.2 µl of distilled water. After a 5-min denaturation at 95°C, the amplification of target cDNA was performed for 40-50 cycles according to the following steps: denaturation at 95°C (0 s), annealing at 60°C (10 s), and elongation at 72°C (40 s). After each elongation step, the SYBR Green fluorescence was measured either immediately at 72°C (Mrp1 and 6) or after the temperature was raised to 85°C to prevent a contribution of primer dimers (Mrp3). At the end of the PCR, a melting curve analysis was performed by gradually increasing the temperature from 72-95°C (0.1°C/s). Moreover, at the end of some experiments, RT-PCR products were removed from capillaries and analyzed by gel electrophoresis to confirm the presence and assess the purity of the amplicons of interest. After PCR was completed, the SYBR Green fluorescent signal was analyzed and converted into a relative number of copies of target molecules. For this purpose, the results of a series of standards prepared by successive dilutions and plotted against the logarithm of the concentration were used to estimate the relative amount of specific mRNA initially present in the various samples.
Indirect immunofluorescence microscopy.
After rapid perfusion with ice-cold 0.9% NaCl for 1 min, the liver was
frozen in isopentane precooled in liquid nitrogen and stored at
80°C. Tissue sections (5 µm) were prepared with a microtome (Carl
Zeiss, Thornwood, NY), air-dried for 2 h, and fixed with acetone
at
20°C for 10 min. In some experiments related to Mrp3, livers
were fixed by perfusion with 4% paraformaldehyde for 8 min, followed
by cryosection. After incubation with PBS for 5 min, sections were
incubated with 3% donkey serum in PBS containing 0.03% Triton X-100
for 30 min to block nonspecific sites. Sections were incubated with the
primary antibodies (1:50-1:200) for 2 h followed by three
washes with PBS. Then, Cy2- or Cy3-conjugated donkey anti-mouse IgG
(Jackson ImmunoResearch Laboratory, West Grove, PA) (1:100) was
applied to visualize the fluorescence. After another four extensive
washes with PBS, sections were mounted in Vectashield mounting medium
(Vector Laboratories, Burlingame, CA) and studied in a confocal
scanning laser microscope (True Confocal Scanner Leica TCS SP II).
Sections from control and pregnant rats were treated in parallel in all
steps in the same slides in the same well. In the present study, the
acquired images are not appropriate for quantitation, because settings
(gain, contrast, Z-position, or horizontal level of the specimen in
which the images were recorded) of the confocal laser scanning
microscope were not exactly the same for different tissue sections.
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RESULTS |
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Hepatic Mrp2 expression and localization in pregnancy.
Previous studies showed that in pregnancy, Mrp2 protein in liver plasma
membrane was significantly decreased, whereas its mRNA expression was
stable (6), indicating posttranscriptional regulation of
Mrp2 in pregnancy. However, relocalization of Mrp2 into subapical
vesicles might also lead to changes in its detection in plasma membrane
by Western analysis. To clarify this issue, we examined the expression
of Mrp2 in total liver homogenate in control and pregnant rats. As
shown in Fig. 1, left, Mrp2
protein expression in total liver homogenate also decreased to 50% of control levels, consistent with the decreased expression of Mrp2 in
plasma membrane (6). Protein levels of Oatp1 and Oatp2, the transporters responsible for the hepatic uptake of organic anions
in basolateral membrane, were also examined in whole liver homogenate.
As shown in Fig. 1, right, Oatp1 expression did not change,
whereas Oatp2 protein expression was significantly decreased by 45% in
pregnancy.
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Hepatic Mrp1 and Mrp3 mRNA expression in pregnant rats.
We then examined the effects of pregnancy on the expression and
localization of Mrp1 and Mrp3. mRNA levels of transporters were
analyzed using real-time PCR. The fluorescence signal produced by SYBR
Green I, which is detectable only when inserted in double-strand DNA
and is proportional to the accumulated PCR product, was monitored on-line continuously. The melting curve analysis was performed to
confirm PCR product identity after the completion of amplification as
described in MATERIALS AND METHODS. A typical experiment
detecting Mrp1 mRNA expression is shown in Fig.
3, A-E. A
noise band was manually set to record only the signals significantly
above the background and from the linear part of each amplification
curve (Fig. 3A). The number of cycles was calculated by
determination of the intercept of the linear part of the curve
(identified by the four black crosses) with the threshold line
(indicated by the horizontal green line), represented by the red
crosses. This number of cycles was then plotted against the log
concentrations, or the copy numbers, of target cDNA. A relative
standard curve was then established by using a series of successive
dilutions of specific cDNA produced from a control rat liver (Fig. 3,
A and B). The relative concentration of specific
cDNA in unknown samples, which were amplified together with standards,
was calculated according to the produced standard curve (Fig.
3C). A DNA melting curve was obtained at the end of each
amplification by slowly increasing the temperature from 72 to 95°C
and continuously monitoring the fluorescence and was used to confirm
the specificity of the PCR products. As shown in Fig. 3D,
all samples except for the negative control H2O or RT()
displayed a similar phase transition at ~84°C. In contrast, the
negative control sample (H2O) or RT(
) showed a transition
at lower temperatures, reflecting the presence of unspecific DNA
products, probably primer-dimers. We also determined by gel
electrophoresis that the PCR products obtained on the LightCycler were
composed of a single band of the expected size (Fig. 3E).
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Mrp1 and Mrp3 expression and localization in liver in pregnant
rats.
Mrp1 protein expression did not change, whereas Mrp3 expression
decreased by 50% in pregnancy (Fig. 5).
Mrp3 showed a basolateral immunofluorescent staining in both control
and pregnant rats, which was more evident in those hepatocytes
surrounding the central vein (Fig. 6).
Although we detected Mrp1 in total liver homogenate by Western
analysis, we were not able to distinguish Mrp1 signals from the
background by using confocal immunohistochemistry.
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Hepatic transport of DNP-SG in pregnancy.
An isolated perfused liver experiment was performed to characterize the
effect of pregnancy on transport activity of an organic anion, DNP-SG.
CDNB (30 µM) is hydrophobic and able to diffuse rapidly into
hepatocytes where it is rapidly conjugated with glutathione to form
DNP-SG. DNP-SG is predominantly transported across the canalicular membrane by Mrp2; it is also a substrate for Mrp1 and Mrp3.
Bile flow (µl · min1 · g
1
liver) was lower in pregnant rats throughout the experiment (Fig. 7A), consistent with previous
reports (30). The administration of CDNB induced a
transient increase of bile flow in both groups (Fig. 7A),
which was probably due to the osmotic effect of DNP-SG in bile. The
DNP-SG concentration in bile was decreased slightly but significantly
at some time points (Fig. 7B), whereas the biliary secretion
of DNP-SG was markedly decreased in pregnant rats (Fig. 7C).
In contrast, secretion of DNP-SG
(nmol · min
1 · g
1 liver)
across the basolateral membrane was similar in control and pregnant
rats (Fig. 7D).
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DISCUSSION |
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Hepatic Mrp2 protein expression was significantly decreased in liver homogenate by 50% in pregnant rats, consistent with studies using plasma membrane (6). These studies confirmed that the downregulation in Mrp2 in plasma membrane is due to a decrease in hepatic Mrp2 and not to its relocalization into other cellular compartments. Such a downregulation of Mrp2 was reflected by decreased biliary excretion of DNP-SG, a typical substrate of Mrp2, in pregnancy (Fig. 7). These findings also agree with early studies where biliary excretion of the glucuronide conjugate of the major primary metabolite of phenytoin, 5-phenyl-5-(p-hydroxyphenyl) hydantoin, was significantly decreased in pregnant rats (48). Because Mrp2 mRNA expression is stable in pregnancy versus control rats (6), we postulated that there must be either reduced synthesis or accelerated breakdown of Mrp2 in pregnancy in rats. However, little information is yet available regarding the posttranscriptional regulation of Mrp2.
Downregulation of hepatic Mrp2 in canalicular membrane by CBDL or LPS
treatment appears to result in a reciprocal or compensating increased
expression of the basolateral isoforms Mrp1 or Mrp3 (9, 15, 45,
50). These adaptive responses of transporter expression during
cholestatic liver injury may serve as a compensatory mechanism to
minimize the hepatocellular accumulation of toxic biliary constituents
(9, 45, 47). These studies and findings that
pregnancy decreases elimination of glucuronide conjugate of the
hydroxylated metabolite of phenytoin from the blood (48) led us to examine the expression pattern of Mrp1 and 3 in pregnancy. Increased Mrp3 expression in TR rats and after CBDL has
been attributed to hepatic accumulation of Mrp2 substrates in the
absence or decreased expression of Mrp2 in these models. However, the
expression of hepatic basolateral Mrp1 and Mrp3 was not increased under
the physiological conditions of pregnancy, although the function and
expression of Mrp2 were decreased by 50%. Mrp1 expression was stable,
whereas Mrp3 expression decreased by 50% in livers from pregnant rats.
These findings indicate that the isoforms of Mrps are differentially
regulated in pregnancy and that decreased expression of canalicular
Mrp2 does not necessarily lead to a reciprocal increased expression of
basolateral Mrp1 and Mrp3. Had there been no change in Mrp3 expression
in pregnancy, it would have suggested a threshold below which Mrp2
expression must fall before expression of Mrp1 and 3 are increased. The
present data indicate a more complex regulation of Mrp3 expression.
Mrp3 appears to be transcriptionally downregulated in pregnancy,
because Mrp3 mRNA and protein expression decreased in parallel. Further
studies are needed to identify the factors regulating Mrp3 expression
in pregnancy and in other models where Mrp2 expression is decreased.
Endocytic retrieval of Mrp2 from the canalicular membrane into the subapical space has been found in other experimental models of cholestasis including CBDL, LPS-, phalloidin-, and E217G-induced cholestasis (27, 33, 42, 47). The proper cellular localization of transporters is essential for their transporting activities. The present study shows that the localization of Mrp2 and 3 in hepatocyte did not change in pregnant rat liver. Mrp2 was localized and confined to the canalicular membrane in both pregnant and control rat liver, whereas Mrp3 showed a basolateral immunofluorescent staining in both control and pregnant rats, which was more evident in those hepatocytes surrounding the central vein (Figs. 2 and 6).
By using isolated perfused liver, the present study also describes the
functional changes in Mrp2-mediated transport of DNP-SG across the
canalicular membrane. Early studies showed that the content of liver
glutathione does not change, whereas
glutathione-S-transferase activity for CDNB increases during
pregnancy (8, 34, 37). Because the enzyme activity is
20,000-40,000
nmol · min1 · g
1 liver,
which is much higher than Mrp2 transport activity across the
canalicular membrane (maximal 70 nmol · min
1 · g
1 liver) in
the present study, the limiting step for DNP-SG secretion into bile is
transport and not conjugation. Therefore, the effects of changes in
metabolism of CDNB in pregnancy are almost certainly negligible. A
previous study (1) using dibromosulfophthalein, an Mrp2
substrate that is directly transported to bile with no conjugation in
liver, also showed a significantly decreased biliary excretion in
pregnant rats. Initially we anticipated a decreased biliary efflux and
a greater efflux of DNP-SG across the basolateral membrane in pregnant
rat liver. Consistent with the decreased expression of Mrp2, the
biliary secretion of DNP-SG was significantly decreased in pregnant
rats (Fig. 7C). The DNP-SG concentrations in bile were
slightly but significantly decreased at some time points in pregnant
rat liver, although the decrease was small relative to the decrease in
bile flow (Fig. 7, A and B). The decrease in bile
flow is likely due, in part, to decreased Mrp2 expression, because
Mrp2-mediated transport of glutathione is considered a major
contribution to bile flow (2). Although expression of Bsep
is not changed in pregnancy, the maximal secretory rate for TC is
significantly decreased in pregnant rat liver (6,
30). Taken together, these data suggest that Mrp2
function is decreased to a slightly greater extent than is Bsep
function or that of any other transporters that mediate transport of
osmotically active solutes that contribute to bile flow. In
phalloidin-induced cholestasis, the LTC4 secretion rate,
but not its bile concentration, is significantly decreased
(42), leading the authors to estimate that Mrp2 function is impaired in parallel with other mechanisms contributing to bile
flow. In E217G-induced cholestasis, both DNP-SG
concentration in bile and its biliary secretion rate are decreased
(33), whereas the concentration of bile salt is increased
(32, 49). The data imply that Mrp2 function is impaired to
a greater extent than that of bile salt or other transporters involved
in bile formation in E217G-induced cholestasis. Therefore,
depending on the cholestatic model, the concentration of a solute in
bile may be disassociated from its excretory rate. In contrast,
secretion of DNP-SG across the basolateral membrane was similar in
control and pregnant rats (Fig. 7D). The similar
basolateral efflux rates of DNP-SG in control and pregnancy in the
face of its decreased biliary excretion could be due to the decreased
expression of Oatp2 and Mrp3 in pregnancy. Further studies are needed
to determine the factors and transporters that regulate basolateral
efflux in pregnancy.
Mrp1 expression is very low in normal hepatocytes, and its expression in hepatocytes is related to proliferation (40, 41). In pregnant rats, liver mass is increased 30-40% and is at least partly due to an increase in the number of hepatocytes (data not shown). Although the extent of hepatocyte proliferation in pregnancy is poorly documented, it is clear that Mrp1 expression did not change.
We (6) have shown that Oatp2 mRNA expression is significantly decreased in pregnancy, whereas the decrease in Oatp2 protein expression in plasma membrane did not reach statistical significance. In the present study, we observed a significant decrease of Oatp2 protein in total liver homogenate in pregnancy (Fig. 1, right), which agrees with the decreased uptake of E217G in hepatocytes from pregnant rats (5). The downregulation of Oatp2 in pregnancy would result in diminished uptake of organic anions. Decreased hepatic extraction of Oatp2 substrates could serve to protect the liver from hepatotoxins. Conversely, decreased hepatic extraction of dietary constituents would increase exposure of the maternal and fetal organism to potentially toxic agents. It is not known whether other secretory organs such as the kidney could play a compensatory role in such a situation. The lack of change of Oatp1 expression also indicates differential regulation of this family of transporters.
In summary, in contrast to other experimental models of cholestasis, the expression of basolateral Mrp1 and Mrp3 in rat liver was not induced in pregnancy compared with controls, although the function and expression of Mrp2 was markedly decreased. Rather, the expression of Mrp3 was significantly decreased in liver of pregnant rats.
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ACKNOWLEDGEMENTS |
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We thank Drs. Hiroshi Suzuki and Yuichi Sugiyama, Tokyo, Japan for their generous provision of Mrp3 antibody.
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FOOTNOTES |
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This work was supported by Public Health Service Grant GM-55343.
Address for reprint requests and other correspondence: M. Vore, H.S.R.B. 306, Graduate Center for Toxicology, Univ. of Kentucky, 800 Rose St., Lexington, KY 40536-0305 (E-mail: maryv{at}uky.edu).
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.
May 15, 2002;10.1152/ajpgi.00126.2002
Received 1 April 2002; accepted in final form 14 May 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Auansakul, AC,
and
Vore M.
The effect of pregnancy and estradiol-17 beta treatment on the biliary transport maximum of dibromosulfophthalein, and the glucuronide conjugates of 5-phenyl-5-p-hydroxyphenyl[14C]hydantoin and [14C]morphine in the isolated perfused rat liver.
Drug Metab Dispos
10:
344-349,
1982[Abstract].
2.
Ballatori, N,
and
Truong AT.
Glutathione as a primary osmotic driving force in hepatic bile formation.
Am J Physiol Gastrointest Liver Physiol
263:
G617-G624,
1992
3.
Bera, TK,
Lee S,
Salvatore G,
Lee B,
and
Pastan I.
MRP8, a new member of ABC transporter superfamily, identified by EST database mining and gene prediction program, is highly expressed in breast cancer.
Mol Med
7:
509-516,
2001[ISI][Medline].
4.
Borst, P,
Evers R,
Kool M,
and
Wijnholds J.
A family of drug transporters: the multidrug resistance-associated proteins.
J Natl Cancer Inst
92:
1295-1302,
2000
5.
Brock, WJ,
and
Vore M.
The effect of pregnancy and treatment with 17 -estradiol on the transport of organic anions into isolated rat hepatocytes.
Drug Metab Dispos
12:
713-716,
1984[Abstract].
6.
Cao, J,
Huang L,
Liu Y,
Hoffman T,
Stieger B,
Meier PJ,
and
Vore M.
Differential regulation of hepatic bile salt and organic anion transporters in pregnant and postpartum rats and the role of prolactin.
Hepatology
33:
140-147,
2001[ISI][Medline].
7.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
8.
Di Ilio, C,
Sacchetta P,
Del Boccio G,
Muccini A,
and
Polidoro G.
Glutathione-related enzyme activities in pregnant rat liver.
Experientia
41:
66-67,
1985[ISI][Medline].
9.
Donner, MG,
and
Keppler D.
Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver.
Hepatology
34:
351-359,
2001[ISI][Medline].
10.
Eckhardt, U,
Schroeder A,
Stieger B,
Hochli M,
Landmann L,
Tynes R,
Meier PJ,
and
Hagenbuch B.
Polyspecific substrate uptake by the hepatic organic anion transporter Oatp1 in stably transfected CHO cells.
Am J Physiol Gastrointest Liver Physiol
276:
G1037-G1042,
1999
11.
Evers, R,
Kool M,
van Deemter L,
Janssen H,
Calafat J,
Oomen LC,
Paulusma CC,
Oude Elferink RP,
Baas F,
Schinkel AH,
and
Borst P.
Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA.
J Clin Invest
101:
1310-1319,
1998
12.
Gotoh, Y,
Suzuki H,
Kinoshita S,
Hirohashi T,
Kato Y,
and
Sugiyama Y.
Involvement of an organic anion transporter (canalicular multispecific organic anion transporter/multidrug resistance-associated protein 2) in gastrointestinal secretion of glutathione conjugates in rats.
J Pharmacol Exp Ther
292:
433-439,
2000
13.
Hagenbuch, B,
Stieger B,
Foguet M,
Lubbert H,
and
Meier PJ.
Functional expression cloning and characterization of the hepatocyte Na+/bile acid cotransport system.
Proc Natl Acad Sci USA
88:
10629-10633,
1991[Abstract].
14.
Hinchman, CA,
Matsumoto H,
Simmons TW,
and
Ballatori N.
Intrahepatic conversion of a glutathione conjugate to its mercapturic acid. Metabolism of 1-chloro-2,4-dinitrobenzene in isolated perfused rat and guinea pig livers.
J Biol Chem
266:
22179-22185,
1991
15.
Hirohashi, T,
Suzuki H,
Ito K,
Ogawa K,
Kume K,
Shimizu T,
and
Sugiyama Y.
Hepatic expression of multidrug resistance-associated protein-like proteins maintained in eisai hyperbilirubinemic rats.
Mol Pharmacol
53:
1068-1075,
1998
16.
Hirohashi, T,
Suzuki H,
and
Sugiyama Y.
Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3).
J Biol Chem
274:
15181-15185,
1999
17.
Hopper, E,
Belinsky MG,
Zeng H,
Tosolini A,
Testa JR,
and
Kruh GD.
Analysis of the structure and expression pattern of MRP7 (ABCC10), a new member of the MRP subfamily.
Cancer Lett
162:
181-191,
2001[ISI][Medline].
18.
Ito, K,
Suzuki H,
Hirohashi T,
Kume K,
Shimizu T,
and
Sugiyama Y.
Functional analysis of a canalicular multispecific organic anion transporter cloned from rat liver.
J Biol Chem
273:
1684-1688,
1998
19.
Jacquemin, E,
Hagenbuch B,
Stieger B,
Wolkoff AW,
and
Meier PJ.
Expression cloning of a rat liver Na+-independent organic anion transporter.
Proc Natl Acad Sci USA
91:
133-137,
1994[Abstract].
20.
Jedlitschky, G,
Burchell B,
and
Keppler D.
The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides.
J Biol Chem
275:
30069-30074,
2000
21.
Jedlitschky, G,
Leier I,
Buchholz U,
Barnouin K,
Kurz G,
and
Keppler D.
Transport of glutathione, glucuronate, and sulfate conjugates by the MRP gene-encoded conjugate export pump.
Cancer Res
56:
988-994,
1996[Abstract].
22.
Jedlitschky, G,
Leier I,
Buchholz U,
Center M,
and
Keppler D.
ATP-dependent transport of glutathione S-conjugates by the multidrug resistance-associated protein.
Cancer Res
54:
4833-4836,
1994[Abstract].
23.
Kao, H,
Huang J,
and
Chang M.
cDNA cloning and genomic organization of the murine MRP7, a new ATP-binding cassette transporter.
Gene
286:
299-306,
2002[ISI][Medline].
24.
Keppler, D,
and
Konig J.
Hepatic canalicular membrane 5: expression and localization of the conjugate export pump encoded by the MRP2 (cMRP/cMOAT) gene in liver.
FASEB J
11:
509-516,
1997
25.
Keppler, D,
and
Konig J.
Hepatic secretion of conjugated drugs and endogenous substances.
Semin Liver Dis
20:
265-272,
2000[ISI][Medline].
26.
Kool, M,
de Haas M,
Scheffer GL,
Scheper RJ,
van Eijk MJ,
Juijn JA,
Baas F,
and
Borst P.
Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines.
Cancer Res
57:
3537-3547,
1997[Abstract].
27.
Kubitz, R,
Wettstein M,
Warskulat U,
and
Haussinger D.
Regulation of the multidrug resistance protein 2 in the rat liver by lipopolysaccharide and dexamethasone.
Gastroenterology
116:
401-410,
1999[ISI][Medline].
28.
Kullak-Ublick, GA,
Beuers U,
and
Paumgartner G.
Hepatobiliary transport.
J Hepatol
32, Suppl1:
3-18,
2000[Medline].
29.
Leier, I,
Jedlitschky G,
Buchholz U,
and
Keppler D.
Characterization of the ATP-dependent leukotriene C4 export carrier in mastocytoma cells.
Eur J Biochem
220:
599-606,
1994[Abstract].
30.
Liu, Y,
Hyde JF,
and
Vore M.
Prolactin regulates maternal bile secretory function post partum.
J Pharmacol Exp Ther
261:
560-566,
1992[Abstract].
31.
Madon, J,
Hagenbuch B,
Landmann L,
Meier PJ,
and
Stieger B.
Transport function and hepatocellular localization of mrp6 in rat liver.
Mol Pharmacol
57:
634-641,
2000
32.
Meyers, M,
Slikker W,
Pascoe G,
and
Vore M.
Characterization of cholestasis induced by estradiol-17 -D-glucuronide in the rat.
J Pharmacol Exp Ther
214:
87-93,
1980[Abstract].
33.
Mottino, AD,
Cao J,
Veggi LM,
Crocenzi F,
Roma MG,
and
Vore M.
Altered localization and activity of canalicular Mrp2 in estradiol-17--D-glucuronide-induced cholestasis.
Hepatology
35:
1409-1419,
2002[ISI][Medline].
34.
Neish, WJ,
and
Key L.
Polyamines and glutathione in livers of normal rats of different ages and in livers of pregnant rats.
Biochem Pharmacol
17:
497-502,
1968[ISI][Medline].
35.
Noe, B,
Hagenbuch B,
Stieger B,
and
Meier PJ.
Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain.
Proc Natl Acad Sci USA
94:
10346-10350,
1997
36.
Ogawa, K,
Suzuki H,
Hirohashi T,
Ishikawa T,
Meier PJ,
Hirose K,
Akizawa T,
Yoshioka M,
and
Sugiyama Y.
Characterization of inducible nature of MRP3 in rat liver.
Am J Physiol Gastrointest Liver Physiol
278:
G438-G446,
2000
37.
Polidoro, G,
Di Ilio C,
Arduini A,
and
Federici G.
Effect of pregnancy on hepatic glutathione S-transferase activities in the rat.
Biochem Pharmacol
30:
1859-1860,
1981[ISI][Medline].
38.
Reichel, C,
Gao B,
Van Montfoort J,
Cattori V,
Rahner C,
Hagenbuch B,
Stieger B,
Kamisako T,
and
Meier PJ.
Localization and function of the organic anion-transporting polypeptide Oatp2 in rat liver.
Gastroenterology
117:
688-695,
1999[ISI][Medline].
39.
Roelofsen, H,
Hooiveld GJ,
Koning H,
Havinga R,
Jansen PL,
and
Muller M.
Glutathione S-conjugate transport in hepatocytes entering the cell cycle is preserved by a switch in expression from the apical MRP2 to the basolateral MRP1 transporting protein.
J Cell Sci
112:
1395-1404,
1999
40.
Roelofsen, H,
Muller M,
and
Jansen PL.
Regulation of organic anion transport in the liver.
Yale J Biol Med
70:
435-445,
1997[ISI][Medline].
41.
Roelofsen, H,
Vos TA,
Schippers IJ,
Kuipers F,
Koning H,
Moshage H,
Jansen PL,
and
Muller M.
Increased levels of the multidrug resistance protein in lateral membranes of proliferating hepatocyte-derived cells.
Gastroenterology
112:
511-521,
1997[ISI][Medline].
42.
Rost, D,
Kartenbeck J,
and
Keppler D.
Changes in the localization of the rat canalicular conjugate export pump Mrp2 in phalloidin-induced cholestasis.
Hepatology
29:
814-821,
1999[ISI][Medline].
43.
Schuetz, JD,
Connelly MC,
Sun D,
Paibir SG,
Flynn PM,
Srinivas RV,
Kumar A,
and
Fridland A.
MRP4: a previously unidentified factor in resistance to nucleoside-based antiviral drugs.
Nat Med
5:
1048-1051,
1999[ISI][Medline].
44.
Sokolovsky, MST,
and
Patchornik A.
Nonenzymatic cleavages of peptide chains at the cysteine and serine residues through their conversion to dehydroalanine (DHAL). II. The specific chemical cleavage of cysteinyl peptides.
J Am Chem Soc
86:
1210-1217,
1964.
45.
Soroka, CJ,
Lee JM,
Azzaroli F,
and
Boyer JL.
Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver.
Hepatology
33:
783-791,
2001[ISI][Medline].
46.
Suzuki, H,
and
Sugiyama Y.
Excretion of GSSG and glutathione conjugates mediated by MRP1 and cMOAT/MRP2.
Semin Liver Dis
18:
359-376,
1998[ISI][Medline].
47.
Trauner, M,
Arrese M,
Soroka CJ,
Ananthanarayanan M,
Koeppel TA,
Schlosser SF,
Suchy FJ,
Keppler D,
and
Boyer JL.
The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive cholestasis.
Gastroenterology
113:
255-264,
1997[ISI][Medline].
48.
Vore, M,
Soliven E,
and
Blunden M.
The effect of pregnancy on the biliary excretion of [14C]phenytoin in the rat.
J Pharmacol Exp Ther
208:
257-262,
1979[Abstract].
49.
Vore, M,
and
Brouwer KLR
Cholestatic properties of steroid glucuronides.
In: Toxicology of the Liver (2nd ed.), edited by Plaa GL,
and Hewitt WR.. Washington, DC: Taylor & Frances, 1998, p. 323-346.
50.
Vos, TA,
Hooiveld GJ,
Koning H,
Childs S,
Meijer DK,
Moshage H,
Jansen PL,
and
Muller M.
Up-regulation of the multidrug resistance genes, Mrp1 and Mdr1b, and down-regulation of the organic anion transporter, Mrp2, and the bile salt transporter, Spgp, in endotoxemic rat liver.
Hepatology
28:
1637-1644,
1998[ISI][Medline].
51.
Wijnholds, J,
Mol CA,
van Deemter L,
de Haas M,
Scheffer GL,
Baas F,
Beijnen JH,
Scheper RJ,
Hatse S,
De Clercq E,
Balzarini J,
and
Borst P.
Multidrug-resistance protein 5 is a multispecific organic anion transporter able to transport nucleotide analogs.
Proc Natl Acad Sci USA
97:
7476-7481,
2000