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
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-β-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).
MATERIALS AND METHODS
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).
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).
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 · min−1 · 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.
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% β-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.
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.
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.
Immunoflouroscence studies by confocal microscopy indicated no change in the pattern of tissue localization of Mrp2 in pregnancy (Fig.2). Mrp2 was localized and confined to the canalicular membrane in both pregnant and control rat liver.
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. 3 A). 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.3 C). 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. 3 D, 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. 3 E).
The real-time PCR experiments revealed that Mrp1 mRNA expression did not change, whereas Mrp3 mRNA and Mrp6 mRNA expression significantly decreased by 40 and 60%, respectively, in pregnancy relative to controls (Fig. 4). To minimize errors owing to variations occurring during RNA extraction and quantification, the results were normalized to the amount of mRNA coding in the same sample for β-actin, a housekeeping gene.
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.
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 · min−1 · g−1liver) was lower in pregnant rats throughout the experiment (Fig.7 A), consistent with previous reports (30). The administration of CDNB induced a transient increase of bile flow in both groups (Fig. 7 A), 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. 7 B), whereas the biliary secretion of DNP-SG was markedly decreased in pregnant rats (Fig. 7 C). In contrast, secretion of DNP-SG (nmol · min−1 · g−1 liver) across the basolateral membrane was similar in control and pregnant rats (Fig. 7 D).
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. 2and 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 · min−1 · 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. 7 C). 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. 7 D). 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.
We thank Drs. Hiroshi Suzuki and Yuichi Sugiyama, Tokyo, Japan for their generous provision of Mrp3 antibody.
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:).
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
- Copyright © 2002 the American Physiological Society