Localization Of Mrp2 Research Articles

Decreased Mrp2-Dependent Bile Flow in the Post-Warm Ischemic Rat Liver

The link between microcirculatory disturbance and hepatocellular dysfunction remains unknown. The present study was designed to examine the key event of warm ischemia reperfusion (WIR) injury with subsequent cholestasis. A left lobar 70% ischemia and reperfusion rat model was used in this study. The portal vein and hepatic artery to the left lateral lobe of the liver were subjected to 20 min of warm ischemia followed by 60 min of reperfusion to collect bile and to measure its constituents. The hepatocellular injury was increased significantly in livers exposed to WIR, as judged by serum alanine aminotransferase. This event coincided with decreased bile production and biliary concentration of glutathione (GSH), suggesting impaired bile salts-independent bile flow, while biliary phospholipids and bile salts were not decreased. Additionally, hepatic adenosine triphosphate and GSH were not decreased after WIR. Since the biliary GSH, which is a major driving force for bile salts-independent bile flow, is excreted from hepatocytes into the bile via multidrug resistance protein 2 (Mrp2), we examined whether intracellular localization of Mrp2 occurred. Immunohistochemical analyses revealed hepatocellular Mrp2 was retrieved from bile canalicular membrane into the pericanalicular cytoplasm in the post-warm ischemic livers. Microcirculatory disturbance in livers exposed to 20 min of warm ischemia improved to levels comparable to controls. Mrp2 internalization, observed in this study, may play an important determinant of cholestasis in the post-warm ischemic livers.

Expression of the multidrug resistance proteins MRP2 and MRP3 in human cholangiocellular carcinomas

Cholangiocellular carcinomas and gallbladder carcinomas are highly aggressive tumours with a poor prognosis and are generally regarded as chemoresistant tumours. Overexpression of ATP-binding cassette transporters of the multidrug resistance protein (MDR) and multidrug resistance-related protein (MRP) family in cancer cells is a major cause for the multidrug resistance phenotype in vitro and in vivo. To further define the role of MRP family members in biliary tract cancer, we studied the expression and localization of MRP2 and MRP3 in cholangiocellular carcinomas and gallbladder carcinomas. The expression and cellular localization of the multidrug resistance proteins MRP2 and MRP3 in human cholangiocellular carcinomas and gallbladder carcinomas were analysed by immunohistochemistry using isoform-specific antibodies. Expression of MRP isoforms was studied in vitro in Mz-ChA-1 cells derived from gallbladder adenocarcinoma by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting and immunofluorescence microscopy. Mz-ChA-1 cells constitutively expressed MDR P-glycoproteins, MRP1, MRP2 and MRP3 by RT-PCR, immunoblotting and immunofluorescence microscopy. MRP2 and MRP3 are expressed in the respective apical and basolateral membrane domains. MRP3 was the predominant MRP isoform in gallbladder carcinomas (93%) and cholangiocellular carcinomas (57%), whereas MRP2 expression was detected in only 29% of gallbladder carcinomas and was undetectable in cholangiocellular carcinomas. Our findings suggest that the intrinsic multidrug resistance of cholangiocellular and gallbladder carcinomas seems to be independent of MRP2 expression while the expression of MRP3 may contribute to the MDR phenotype.

The role of radixin in altered localization of canalicular conjugate export pump Mrp2 in cholestatic rat liver

Cholestasis has been associated with the endocytic retrieval of multidrug resistance protein 2 (Mrp2), but its mechanism is still unclear. Recent studies have indicated that radixin, a cross-linker between the actin filaments and membrane proteins, may be activated by phosphorylation and may be required for the canalicular localization of Mrp2. We investigated the role of radixin in the altered localization of Mrp2 in rat models of intrahepatic (ethinyl estradiol treatment) and extrahepatic (bile duct ligation) cholestasis using immunofluorescence microscopy. The changes in localization and expression were analyzed using Scion Image for Windows. In both models, Mrp2 was localized outside as well as inside the ZO-1 staining, indicating partial dislocation from the canalicular membrane. In contrast to the steep elevation of the immunostaining intensity curves for Mrp2 in the controls, the corresponding curves in both models were broadened and flattened, confirming endocytic retrieval into the hepatocytes. Mrp2 and radixin were colocalized at the canalicular domain in the controls, whereas in both cholestatic rats they were dissociated at some canaliculi, indicating the disturbed colocalization of Mrp2 and radixin in cholestasis. The fluorescence of phosphorylated radixin, an active form of radixin, markedly decreased in both cholestatic models, which was supported by the reduced peak fluorescence intensities. The disturbed colocalization of Mrp2 and radixin may contribute to the endocytic retrieval of Mrp2 in cholestasis due to the failure to anchor Mrp2 in the canalicular membrane, in which the phosphorylated radixin may play a major role.

Disruption of function and localization of tight junctional structures and Mrp2 in sustained estradiol-17β-d-glucuronide-induced cholestasis

Estradiol-17beta-D-glucuronide (E(2)17G) induces immediate and profound but transient cholestasis in rats when administered as a single bolus dose. Here, we examined the consequence of sustained E(2)17G cholestasis and assessed the function and localization of the tight junctional proteins zonula occludens-1 (ZO-1) and occludin and of the canalicular transporter multidrug resistance-associated protein-2 (Mrp2). An initial dose of E(2)17G (15 mumol/kg iv) followed by five subsequent doses of 7.5 mumol/kg from 60 to 240 min induced a sustained 40-70% decrease in bile flow. Following their biliary retrograde administration, cholera toxin B subunit-FITC or horseradish peroxidase were detected at the sinusoidal domain, indicating opening of the paracellular route; this occurred as early as 15 min after the first dose as well as 15 min after the last dose of E(2)17G, but not following the administration of vehicle in controls. Localization of ZO-1 and occludin was only slightly affected under acute cholestatic conditions but was severely disrupted under sustained cholestasis, with their appearance suggesting a fragmented structure. Endocytic internalization of Mrp2 to the pericanalicular region was apparent 20 min after a single E(2)17G administration; however, Mrp2 was found more deeply internalized and partially redistributed to the basolateral membrane under sustained cholestasis. In conclusion, acute E(2)17G-induced cholestasis increased permeability of the tight junction, while sustained cholestasis provoked a significant redistribution of ZO-1, occludin, and Mrp2 in addition to increased permeability of the tight junction. Altered tight junction integrity likely contributes to impaired bile secretion and may be causally related to changes in Mrp2 localization.

Phloracetophenone-induced choleresis in rats is mediated through Mrp2

Phloracetophenone (2,4,6-trihydroxyacetophenone, THA) is a potent choleretic in the bile fistula rat, although the mechanism is unknown. In the present study, we examined how THA enhances bile secretion. Stepwise infusions of THA (1-4 micromol/min) in the isolated perfused rat liver resulted in an immediate and dose-dependent increase in bile flow (BF), which reached saturation. The increase in BF was not associated with a change in the excretion of bile acids, suggesting that THA stimulated bile acid-independent bile flow. To further define the mechanism, the effect of THA on the excretion of sulfobromophthalein (BSP) and disulfobromophthalein (DBSP), typical multidrug resistance protein-2 (Mrp2) substrates was examined. THA inhibited the biliary excretion of both substrates. Because DBSP is excreted without conjugation to glutathione, in contrast to BSP, the findings suggest that THA might compete with DBSP and BSP metabolites at a common canalicular transport site, presumably Mrp2. THA infusions had no effect on the subcellular localization and distribution of either Mrp2 or the bile salt export pump (Bsep), nor the integrity of the tight junction. In contrast, the choleretic activity of THA was completely absent in the TR(-) rat, an animal model that lacks Mrp2, directly implicating this canalicular export pump as the mechanisms by which THA is excreted in bile. THA also partially reversed the cholestatic effects of estradiol-17beta-D-glucuronide, a process also dependent on Mrp2. In conclusion, the choleretic activity of THA and its possible metabolites is dependent on Mrp2. THA appears to stimulate BF by its osmotic effects and may attenuate the cholestatic effects of hepatotoxins undergoing biotransformation and excretion via similar pathways.

Inchinkoto, a herbal medicine, and its ingredients dually exert Mrp2/MRP2-mediated choleresis and Nrf2-mediated antioxidative action in rat livers

Inchinkoto (ICKT), a herbal medicine, has been recognized in Japan and China as a "magic bullet" for jaundice. To explore potent therapeutic agents for cholestasis, the effects of ICKT or its ingredients on multidrug resistance-associated protein 2 (Mrp2/ MRP2)-mediated choleretic activity, as well as on antioxidative action, were investigated using rats and chimeric mice with livers that were almost completely repopulated with human hepatocytes. Biliary excretion of Mrp2 substrates and the protein mass, subcellular localization, and mRNA level of Mrp2 were assessed in rats after 1-wk oral administration of ICKT or genipin, a major ingredient of ICKT. Administration of ICKT or genipin to rats for 7 days increased bile flow and biliary excretion of bilirubin conjugates. Mrp2 protein and mRNA levels and Mrp2 membrane densities in the bile canaliculi and renal proximal tubules were significantly increased in ICKT- or genipin-treated rat livers and kidneys. ICKT and genipin, thereby, accelerated the disposal of intravenously infused bilirubin. The treatment also increased hepatic levels of heme oxygenase-1 and GSH by a nuclear factor-E2-related factor (Nrf2)-dependent mechanism. Similar effects of ICKT on MRP2 expression levels were observed in humanized livers of chimeric mice. In conclusion, these findings provide the rationale for therapeutic options of ICKT and its ingredients that should potentiate bilirubin disposal in vivo by enhancing Mrp2/MRP2-mediated secretory capacities in both livers and kidneys as well as Nrf2-mediated antioxidative actions in the treatment of cholestatic liver diseases associated with jaundice.

Distribution and functional activity of P-glycoprotein and multidrug resistance-associated proteins in human brain microvascular endothelial cells in hippocampal sclerosis

Multidrug resistance protein, also referred as P-glycoprotein (P-gp, MDR1; ABCB1) and multidrug resistance-associated protein (MRP) 1 ( ABCC1) and 2 ( ABCC2) are, thus far, candidates to cause antiepileptic drug (AED) resistance epilepsy. In this study, we investigated P-gp, MRP1 and MRP2 expression, localization and functional activity on cryosections and isolated human brain-derived microvascular endothelial cells (HBMEC) from epileptic patients (HBMEC-EPI) with hippocampal sclerosis (HS), as compared with HBMEC isolated from normal brain cortex (HBMEC-CTR). We examined the expression and distribution of three transporters, P-gp, MRP1 and MRP2 on two major parts of the resected tissue, the hippocampus and the parahippocampal gyrus (Gph). P-gp showed diffuse expression not only in endothelium but also by parenchymal cells in both the hippocampus and the Gph. MRP1 labeling was observed in parenchymal cells in the Gph. By contrast, MRP2 was mainly found in endothelium of the hippocampus. P-gp and MRP1 expression in the Gph was relatively high in the patient with long-term seizure history. Quantitative RT-PCR analysis of HBMEC revealed that MDR1, MRP1 as well as MRP5 (ABCC5) and MRP6 (ABCC6) were overexpressed in HBMEC-EPI at the mRNA level. HBMEC from both normal and epilepsy groups displayed protein expression of P-gp, whereas MRP1 and MRP2 were seen only in HBMEC-EPI. Accordingly, it is of particular interest that MRP functional activities were observed in HBMEC-EPI, but not in HBMEC-CTR. Our results suggest that complex MDR expression changes not only in the hippocampus but in the Gph may play a role in AED pharmacoresistance in intractable epilepsy patients with mesial temporal lobe epilepsy (MTLE) by altering the permeability of AEDs across the blood–brain barrier (BBB).

VARIABLE EXPRESSION OF MRP2 (ABCC2) IN HUMAN PLACENTA: INFLUENCE OF GESTATIONAL AGE AND CELLULAR DIFFERENTIATION

MRP2 (ABCC2) is an ATP-binding cassette (ABC)-type membrane protein involved in transport of conjugates of various drugs and endogenous compounds. MRP2 has been localized to the apical membrane of syncytiotrophoblasts and is assumed to be involved in diaplacental transfer of the above substances. It has been shown that both genetic and environmental factors can influence MRP2 expression. We therefore investigated whether gestational age, cellular differentiation, and genetic polymorphisms influence expression and localization of MRP2 in 58 human placenta samples. We detected a significant increase of transporter-mRNA with gestational age by quantitative real-time polymerase chain reaction (MRP2 mRNA/18S rRNA ratio x 1000 +/- S.D.; 0.43 +/- 0.13 in early preterms versus 1.18 +/- 0.44 in late preterms versus 2.1 +/- 0.63 in terms; p < 0.05). MRP2 protein followed the mRNA amount as shown by Western blotting (mean relative band intensity +/- S.D.; 0.56 +/- 0.1 versus 0.7 +/- 0.18 versus 0.92 +/- 0.19; early preterms versus terms p < 0.05). In cultured cytotrophoblasts, MRP2 expression increased with differentiation to syncytiotrophoblasts, with a peak on day 2 (MRP2 mRNA/18S rRNA ratio x 1000 +/- S.D.; 0.06 +/- 0.01 versus 0.88 +/- 0.27 versus 0.24 +/- 0.02 on days 0, 2, and 4). Moreover, we studied the effect of single nucleotide polymorphisms (C-24T; G1249A, and C3972T) in the MRP2 gene on placental expression. One of these polymorphisms (G1249A) resulted in a significantly reduced expression of MRP2 mRNA in preterms. In summary, the expression of MRP2 in human placenta is influenced by gestational age, cellular differentiation, and genetic factors.

Treatment of Hyperbilirubinemia in Eisai Hyperbilirubinemic Rat by Transfecting Human MRP2/ABCC2 Gene

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is predominantly expressed in the liver canalicular membrane and plays an important role in the biliary excretion of organic anions including glucuronide and glutathione conjugates. The purpose of this study is to construct a new evaluation system for human MRP2 by expressing human MRP2 in Eisai hyperbilirubinemic rat (EHBR) liver, the rat Mrp2 function of which is hereditarily defective. In order to express human MRP2 in liver, we used the Tet-off adenovirus expression system. After 72 h infection, we evaluated the protein expression and localization in the liver and the transport activity of [(3)H]E(2)17ssG and [(3)H]DNP-SG by preparing canalicular membrane vesicles (CMVs). We also evaluated the biliary excretion and plasma concentration of DBSP after bolus administration and the plasma concentration of endogenous direct and indirect bilirubin. The localization of human MRP2 in EHBR liver was found to be at the bile canalicular membrane. Clear ATP-dependent uptake of [(3)H]E(2)17ssG and [(3)H]DNP-SG into CMVs was observed by using the CMVs prepared from the liver where human MRP2 was transfected. Furthermore, the blood to bile clearance of DBSP increased approximately 3-fold after expression of human MRP2. In addition, the plasma direct bilirubin level in EHBR was reduced by the expression of human MRP2. These results suggest that this evaluation system for human MRP2 may be useful for evaluating the function of human MRP2.

Role of Glycosylation in Trafficking of Mrp2 in Sandwich-Cultured Rat Hepatocytes

The multidrug resistance-associated protein (MRP) family plays a major role in the hepatic excretion of organic anions. The expression, localization, and function of Mrp2 (Abcc2), a canalicular multispecific organic anion transport protein, were studied in sandwich-cultured rat hepatocytes. The amount of Mrp2 protein remained constant in sandwich-cultured rat hepatocytes over 4 days in culture, but the molecular mass increased approximately 10 kDa from 190 to 200 kDa. Mrp2 was internalized initially after hepatocyte isolation and was gradually sorted to the canalicular membrane. Disposition of 5-(6)-carboxy-2',7'-dichlorofluorescein (CDF), an Mrp2 substrate, confirmed the changes in Mrp2 localization. CDF was localized predominantly inside hepatocytes at day 0 and gradually localized to the canalicular domain over time in culture. By day 4 in culture, CDF was localized exclusively in the canalicular networks. Tunicamycin, an inhibitor of glycosylation, decreased the molecular mass and simultaneously impaired the trafficking of Mrp2 to the canalicular membrane. Treatment of lysates from both day 0 (Mrp2, 190 kDa) and day 4 (Mrp2, 200 kDa) sandwich-cultured rat hepatocytes with peptide N-glycosidase F, a deglycosylation agent, resulted in a band of 180 kDa, suggesting that Mrp2 from both day 0 and day 4 was glycosylated, but Mrp2 on day 4 was more glycosylated than on day 0. In conclusion, these data support the hypothesis that glycosylation of Mrp2 is responsible for the increase in molecular mass and may be involved in directing the canalicular localization of Mrp2 in sandwich-cultured rat hepatocytes over days in culture.

Involvement of Mrp2 in Hepatic and Intestinal Disposition of Dinitrophenyl-S-glutathione in Partially Hepatectomized Rats

The ability of the liver and small intestine for secretion of dinitrophenyl-S-glutathione (DNP-SG), a substrate for multidrug resistance-associated protein 2 (Mrp2), into bile and lumen, respectively, as well as expression of Mrp2 in both tissues, were assessed in 70-75% hepatectomized rats. An in vivo perfused intestinal model was used. A single i.v. dose of 30 micromol/kg b.w. of 1-chloro-2,4-dinitrobenzene (CDNB) was administered and its glutathione conjugate, DNP-SG, was determined by HPLC in bile and intestinal perfusate. One and seven days after hepatectomy, biliary excretion of DNP-SG was decreased by 90 and 50% with respect to shams, respectively, when expressed per mass unit. In contrast, intestinal excretion was increased by 63% or unchanged one and seven days post-hepatectomy, respectively. Tissue content of DNP-SG 5 min after CDNB administration was substantially decreased in liver and significantly increased in intestine, one day post-hepatectomy. Western and immunofluorescence studies revealed preserved levels and localization of Mrp2 in both tissues from hepatectomized animals, irrespective of the time analyzed. In spite of preserved expression of Mrp2, the higher availability of DNP-SG in intestinal cells, likely as a consequence of increased glutathione-S-transferase-mediated conjugation of CDNB, may explain the in vivo findings. Further experiments in isolated hepatocytes suggested that decreased synthesis of DNP-SG rather than altered canalicular transport is responsible for the substantial impairment in excretion of this compound into bile. Taken together, these results indicate that the intestine may partially compensate for liver DNP-SG disposition, particularly shortly after surgery, while liver capability is recovering.

Role of microtubules in estradiol-17beta-D-glucuronide-induced alteration of canalicular Mrp2 localization and activity.

Estradiol-17beta-D-glucuronide (E2-17G) induces a marked but reversible inhibition of bile flow in the rat together with endocytic retrieval of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane to intracellular structures. We analyzed the effect of pretreatment (100 min) with the microtubule inhibitor colchicine or lumicholchicine, its inactive isomer (1 micromol/kg iv), on changes in bile flow and localization and function of Mrp2 induced by E2-17G (15 micromol/kg iv). Bile flow and biliary excretion of bilirubin, an endogenous Mrp2 substrate, were measured throughout, whereas Mrp2 localization was examined at 20 and 120 min after E2-17G by confocal immunofluorescence microscopy and Western analysis. Colchicine pretreatment alone did not affect bile flow or Mrp2 localization and activity over the short time scale examined (3-4 h). Administration of E2-17G to colchicine-pretreated rats induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as well as internalization of Mrp2 at 20 min. These alterations were of a similar magnitude as in rats pretreated with lumicolchicine followed by E2-17G. Bile flow and Mrp2 localization and activity were restored to control levels within 120 min of E2-17G in animals pretreated with lumicolchicine. In contrast, in colchicine-pretreated rats followed by E2-17G, bile flow and Mrp2 activity remained significantly inhibited by 60%, and confocal and Western studies revealed sustained internalization of Mrp2 120 min after E2-17G. We conclude that recovery from E2-17G cholestasis, associated with exocytic insertion of Mrp2 in the canalicular membrane, but not its initial E2-17G-induced endocytosis, is a microtubule-dependent process.

Differential multidrug resistance-associated protein 1 through 6 isoform expression and function in human intestinal epithelial Caco-2 cells.

Multidrug resistance-associated protein (MRP) isoforms 1 through 6 mRNA are expressed in the human intestine and Caco-2 cells. In Caco-2 cells, the rank order for mRNA expression was MRP2 > or = MRP6 > MRP4 > or = MRP3 > MRP1 = MRP5. The functional expression of MRP-like activity was quantified as the efflux of the fluorescent probe calcein from confluent, polarized monolayers of Caco-2 cells. Calcein efflux was sensitive to temperature, energy depletion, and the MRP antagonist MK571 [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid]. Calcein efflux across the apical membrane of Caco-2 cells exceeded that across the basolateral by approximately 2-fold, correlating with the apical localization of MRP2 visualized by immunocytochemical staining. T84 cells do not express MRP2 and show a predominance of basolateral calcein efflux over apical efflux. MRP3 was localized by immunocytochemical staining to the basolateral membrane. MRP1 staining was not localized to either membrane domain and MRP5 staining was not detected. Thus, basolateral calcein efflux may reflect a function of MRP3 or MRP4 and 6 inferred by their basolateral localization in other tissues. Basolateral, but not apical, calcein efflux was sensitive to glutathione depletion with buthioninesulfoximine, indicating that whereas MRP2-mediated apical efflux is independent of glutathione, basolateral efflux is glutathione-dependent. Benzbromarone, probenecid, pravastatin, and diclofenac were able to inhibit both apical and basolateral calcein efflux. The apical calcein efflux in Caco-2 cells was selectively sensitive to indomethacin and propranolol, but not verapamil or erythromycin, whereas the converse was observed for basal efflux. The differential pharmacological sensitivity of apical (MRP2) and basolateral calcein efflux provides tools for dissecting MRP isoform functional roles.

Enhanced expression of basolateral multidrug resistance protein isoforms Mrp3 and Mrp5 in rat liver by LPS.

Lipopolysaccharide (LPS) induces hepatocellular down-regulation and endocytic retrieval of multidrug resistance protein 2 (Mrp2, Abcc2). Basolateral Mrp isoforms may compensate for the intracellular metabolic changes in cholestasis. Therefore, the effect of LPS on the zonal localization of Mrp2 and Mrp3 and the expression of Mrp3, Mrp4, Mrp5, and Mrp6 mRNA were investigated in rat liver. In normal rat liver Mrp3 was found in pericentral hepatocytes also expressing glutamine synthetase. In LPS-treated rat liver the decrease in Mrp2 protein was most pronounced in pericentral hepatocytes, with only minor down-regulation in periportal hepatocytes. Conversely, induction of Mrp3 was found in pericentral hepatocytes with a low expression of Mrp2. Furthermore, we found a strong induction of Mrp5 mRNA. Likewise, Mrp6 mRNA was up-regulated, however Mrp6 protein expression was not significantly altered. It is concluded that Mrp3 is inversely regulated to Mrp2 in a zonal pattern and may compensate for the LPS-induced loss of Mrp2 in the perivenous area. Induction of pericentral Mrp3 and up-regulation of Mrp5 mRNA may play an important role in the hepatocellular clearance of cholephilic substances and cyclic nucleotides accumulating after LPS treatment.

Genipin enhances Mrp2 (Abcc2)-mediated bile formation and organic anion transport in rat liver.

Inchin-ko-to (ICKT), an herbal medicine, and its ingredients exert potent choleretic effects by a "bile acid-independent" mechanism. The current study was designed to determine whether ICKT or its ingredients potentiate multidrug resistance-associated protein 2 (Mrp2; Abcc2)-mediated choleresis in vivo. Biliary secretion of Mrp2 substrates and the protein mass, subcellular localization, and messenger RNA (mRNA) level of Mrp2 were assessed in rat liver after infusion of genipin, an intestinal bacterial metabolite of geniposide, a major ingredient of ICKT. The function of Mrp2 was also assessed by the adenosine triphosphate (ATP)-dependent uptake of Mrp2-specific substrates using canalicular membrane vesicles (CMVs) from the liver. Infusion of genipin increased bile flow by 230%. It also increased biliary secretion of bilirubin conjugates and reduced glutathione (GSH) by 513% and 336%, respectively, but did not increase bile acid secretion. The ATP-dependent uptake of estradiol 17-beta-D-glucuronide (E(2)17 beta G; by 265%), leukotriene C4 (LTC(4); by 161%), taurolithocholate-3-sulfate (TLC-3S; by 266%), and methotrexate (MTX; by 234%) was significantly stimulated in the CMVs from the liver. These effects were not observed in Mrp2-deficient rats. Under these conditions, genipin treatment increased the protein mass of Mrp2 in the CMVs but not the mRNA level. In immunoelectron microscopic studies, a marked increase in Mrp2 density in the canalicular membrane (CM) and microvilli was observed in the genipin-treated liver tissue sections when compared with the vehicle-treated liver tissue sections. In conclusion, genipin may enhance the bile acid-independent secretory capacity of hepatocytes, mainly by stimulation of exocytosis and insertion of Mrp2 in the bile canaliculi. ICKT may be a potent therapeutic agent for a number of cholestatic liver diseases.

Changes in the expression and localization of hepatocellular transporters and radixin in primary biliary cirrhosis

Background/Aims : Expression and localization of human hepatocellular transporters and of radixin, cross-linking actin with some membrane transporters, may change in cholestatic liver diseases. Methods : We investigated the uptake transporters OATP2 (SLC21A6), OATP8 (SLC21A8), and NTCP (SLC10A1), the export pumps MRP2 (ABCC2), MRP3 (ABCC3), MRP6 (ABCC6), and P-glycoproteins (ABCB1, ABCB4, ABCB11), and radixin, in non-icteric primary biliary cirrhosis (PBC stages I–III) and control human liver needle-biopsies using immunofluorescence microscopy and semi-quantitative RT-PCR. Results : Expression and localization of all transporters were unchanged in PBC I-II. Immunostaining intensities of uptake transporters decreased in PBC III with a concomitant decrease in mRNA levels. Immunostaining intensities and mRNA levels of export pumps were similar in controls and PBC I–III, however, irregular MRP2 immunostaining suggested redistribution of MRP2 into intracellular structures in PBC III. Areas of irregular MRP2 immunostaining showed largely reduced radixin immunostaining, whereas normal hepatocytes had MRP2 and radixin confined to the canalicular membrane. Disrupted localization of radixin and MRP2 supports the concept that radixin contributes to the canalicular localization of MRP2. Conclusions : Down-regulation of uptake transporters may contribute to the impaired hepatobiliary elimination in advanced PBC, and partially altered localization of MRP2 may reflect the onset of changes leading to icteric PBC.

Adaptive changes in hepatobiliary transporter expression in primary biliary cirrhosis

Background/Aims: Information about alterations of hepatobiliary transporter expression in primary biliary cirrhosis (PBC) could provide important insights into the pathogenesis of cholestasis. This study aimed to determine the expression of hepatobiliary transport systems for bile salts (Na +/taurocholate cotransporter, NTCP; bile salt export pump, BSEP), organic anions (organic anion transporting protein, OATP2; canalicular conjugate export pump, MRP2; basolateral MRP homologue, MRP3), organic cations (canalicular multidrug export pump, MDR1), and phospholipids (canalicular phospholipid flippase MDR3) in livers from patients with advanced stages of PBC. Methods: Transporter mRNA and protein levels were assessed by reverse transcription polymerase chain reaction and Western blot analysis. Tissue distribution of transporters was investigated by immunohistochemistry and immunofluorescence microscopy. Hepatic bile acids were measured by gas chromatography-mass spectrometry. Results: Compared to controls, basolateral uptake systems (NTCP, OATP2) were reduced, canalicular export pumps for bile salts and bilirubin (BSEP, MRP2) were preserved, while canalicular MDR P-glycoproteins (MDR1, MDR3) and the basolateral efflux pump MRP3 were increased in PBC. Double immunofluorescence labeling with a canalicular marker (dipeptidyl peptidase IV) demonstrated proper canalicular localization of BSEP and MRP2 in PBC. OATP2 and MRP2 expression correlated inversely with hepatic levels of hydrophobic bile acids, while positively correlating with hepatic enrichment with ursodeoxycholic acid. Conclusions: Down-regulation of basolateral uptake systems and maintenance/up-regulation of canalicular and basolateral efflux pumps may represent adaptive mechanisms limiting the accumulation of toxic biliary constituents.

Bile formation: Do not ignore the role of plasma membrane-cytoskeleton linking proteins

The ezrin-radixin-moesin (ERM) family of proteins crosslink ac-tin filaments and integral membrane proteins. Radixin (encoded by Rdx) is the dominant ERM protein in the liver of wildtype mice and is concentrated at bile canalicular membranes (BCMs). Here we show that Rdx(-/-) mice are normal at birth, but their serum concentrations of conjugated bilirubin begin to increase gradually around 4 weeks, and they show mild liver injury after 8 weeks. This phenotype is similar to human conjugated hyperbiliru-binemia in Dubin-Johnson syndrome, which is caused by mutations in the multidrug resistance protein 2 (MRP2, gene symbol ABCC2), although this syndrome is not associated with overt liver injury. In wildtype mice, Mrp2 concentrates at BCMs to secrete conjugated bilirubin into bile. In the BCMs of Rdx / mice, Mrp2 is decreased compared with other BCM proteins such as dipeptidyl peptidase IV (CD26) and P-glycoproteins. In vitro binding studies show that radixin associates directly with the car-boxy-terminal cytoplasmic domain of human MRP2. These findings indicate that radixin is required for secretion of conjugated bilirubin through its support of Mrp2 localization at BCMs.

Radixin deficiency causes conjugated hyperbilirubinemia with loss of Mrp2 from bile canalicular membranes.

The ezrin-radixin-moesin (ERM) family of proteins crosslink actin filaments and integral membrane proteins. Radixin (encoded by Rdx) is the dominant ERM protein in the liver of wildtype mice and is concentrated at bile canalicular membranes (BCMs). Here we show that Rdx(-/-) mice are normal at birth, but their serum concentrations of conjugated bilirubin begin to increase gradually around 4 weeks, and they show mild liver injury after 8 weeks. This phenotype is similar to human conjugated hyperbilirubinemia in Dubin-Johnson syndrome, which is caused by mutations in the multidrug resistance protein 2 (MRP2, gene symbol ABCC2), although this syndrome is not associated with overt liver injury. In wildtype mice, Mrp2 concentrates at BCMs to secrete conjugated bilirubin into bile. In the BCMs of Rdx(-/-) mice, Mrp2 is decreased compared with other BCM proteins such as dipeptidyl peptidase IV (CD26) and P-glycoproteins. In vitro binding studies show that radixin associates directly with the carboxy-terminal cytoplasmic domain of human MRP2. These findings indicate that radixin is required for secretion of conjugated bilirubin through its support of Mrp2 localization at BCMs.

Altered localization and activity of canalicular Mrp2 in estradiol-17β- D-glucuronide–induced cholestasis

Estradiol-17β- D-glucuronide (E 217G), an endogenous metabolite of estradiol, induces a potent dose-dependent and reversible inhibition of bile flow in the rat. We analyzed the effect of a single dose of E 217G (15 μmol/kg, intravenously) to female rats on bile flow and the endocytic retrieval and function of the canalicular multidrug resistance-associated protein 2 (Mrp2) and the effect of pretreatment with dibutyryl-cyclic AMP (DBcAMP; 20 μmol/kg) on these measures. Bile flow was maximally inhibited by 85% within 10 minutes of E 217G and returned to 50% and 100% of control levels within 75 and 120 minutes, respectively. Western analysis of total homogenates and mixed plasma and intracellular membranes suggested partial internalization of Mrp2 during the acute phase of cholestasis at 20 minutes and during the period of recovery from cholestasis at 75 minutes, which returned to control levels by 180 minutes after E 217G. Confocal analysis confirmed Western studies and demonstrated endocytic retrieval of Mrp2 from the canalicular membrane into pericanalicular and intracellular domains. The biliary concentration and excretion of the model Mrp2 substrate, dinitrophenyl-S-glutathione (DNP-SG), was impaired in parallel with the extent of Mrp2 retrieval. Pretreatment with DBcAMP partially protected against maximal E 217G cholestasis and the endocytic retrieval and decreased function of Mrp2 at 20 minutes and significantly accelerated the exocytic insertion of Mrp2 into the canalicular membrane and the recovery of bile flow and biliary excretion of DNP-SG. In conclusion, these data indicate that E 217G induces endocytic internalization of Mrp2, which occurs in parallel with decreased bile flow and Mrp2 transport activity. (H EPATOLOGY 2002;35:1409-1419.)