Abcc2 | GeneID:25303 | Rattus norvegicus
[ ] NCBI Entrez Gene
|Gene ID||25303||Official Symbol||Abcc2|
|Full Name||ATP-binding cassette, sub-family C (CFTR/MRP), member 2|
|Description||ATP-binding cassette, sub-family C (CFTR/MRP), member 2|
|Also Known As||Canalicular multispecific organic anion transporter; calicular multispecific organic anion transporter; multidrug resistance associated protein 2|
|Summary||multispecific organic anion transporter; mutation may be responsible for conjugated hyperbilirubinemia in the TR- rat, which is a model for human Dubin-Johnson syndrome [RGD]|
Orthologs and Paralogs
[ ] Monoclonal and Polyclonal Antibodies
|1||abcam||ab15603||MRP2 antibody [M2III-5] (ab15603); Mouse monoclonal [M2III-5] to MRP2|
|2||abcam||ab3373||MRP2 antibody [M2 III-6] (ab3373); Mouse monoclonal [M2 III-6] to MRP2|
|3||abcam||ab50213||MRP2 antibody - Carboxyterminal end (ab50213); Rabbit polyclonal to MRP2 - Carboxyterminal end|
|4||sigma||M8316||Anti-MRP2 antibody produced in rabbit ;|
|GO:0016324||Component||apical plasma membrane|
|GO:0016021||Component||integral to membrane|
|GO:0005887||Component||integral to plasma membrane|
|GO:0042626||Function||ATPase activity, coupled to transmembrane movement of substances|
|GO:0008514||Function||organic anion transmembrane transporter activity|
|GO:0030644||Process||cellular chloride ion homeostasis|
|GO:0015711||Process||organic anion transport|
|GO:0046685||Process||response to arsenic|
|GO:0042493||Process||response to drug|
|GO:0043627||Process||response to estrogen stimulus|
|GO:0009408||Process||response to heat|
|GO:0031427||Process||response to methotrexate|
|GO:0006979||Process||response to oxidative stress|
|GO:0048545||Process||response to steroid hormone stimulus|
- [ ] Grisk O, et al. (2009) "Multidrug resistance-related protein 2 genotype of the donor affects kidney graft function." Pharmacogenet Genomics. 19(4):276-288. PMID:19214140
- [ ] Ban D, et al. (2009) "Decreased Mrp2-dependent bile flow in the post-warm ischemic rat liver." J Surg Res. 153(2):310-316. PMID:18662814
- [ ] Zalups RK, et al. (2009) "MRP2 involvement in renal proximal tubular elimination of methylmercury mediated by DMPS or DMSA." Toxicol Appl Pharmacol. 235(1):10-17. PMID:19063911
- [ ] Qadri I, et al. (2009) "Interaction of hepatocyte nuclear factors in transcriptional regulation of tissue specific hormonal expression of human multidrug resistance-associated protein 2 (abcc2)." Toxicol Appl Pharmacol. 234(3):281-292. PMID:19010343
- [ ] Minami S, et al. (2009) "Posttranslational regulation of Abcc2 expression by SUMOylation system." Am J Physiol Gastrointest Liver Physiol. 296(2):G406-G413. PMID:19074644
- [ ] Itagaki S, et al. (2008) "Contribution of multidrug resistance-associated protein 2 to secretory intestinal transport of organic anions." Biol Pharm Bull. 31(1):146-148. PMID:18175959
- [ ] Rost D, et al. (2008) "Retrieval of the rat canalicular conjugate export pump Mrp2 is associated with a rearrangement of actin filaments and radixin in bile salt-induced cholestasis." Eur J Med Res. 13(7):314-318. PMID:18700187
- [ ] Abe F, et al. (2008) "Involvement of multidrug resistance-associated protein 2 (ABCC2/Mrp2) in biliary excretion of micafungin in rats." Life Sci. 83(7-8):229-235. PMID:18638490
- [ ] Ogura J, et al. (2008) "Alteration of Mrp2 and P-gp expression, including expression in remote organs, after intestinal ischemia-reperfusion." Life Sci. 82(25-26):1242-1248. PMID:18538350
- [ ] MacLean C, et al. (2008) "Closing the gaps: a full scan of the intestinal expression of p-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2 in male and female rats." Drug Metab Dispos. 36(7):1249-1254. PMID:18378562
- [ ] Tanaka Y, et al. (2008) "Ischemia-reperfusion of rat livers decreases liver and increases kidney multidrug resistance associated protein 2 (Mrp2)." Toxicol Sci. 101(1):171-178. PMID:17959626
- [ ] Micuda S, et al. (2008) "Zonation of multidrug resistance-associated protein 2 in rat liver after induction with dexamethasone." J Gastroenterol Hepatol. 23(7 Pt 2):e225-e230. PMID:17683490
- [ ] Zinchuk V, et al. (2007) "Ethanol consumption alters expression and colocalization of bile salt export pump and multidrug resistance protein 2 in the rat." Histochem Cell Biol. 127(5):503-512. PMID:17384956
- [ ] Leslie EM, et al. (2007) "Biotransformation and transport of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in bile duct-cannulated wild-type and Mrp2/Abcc2-deficient (TR ) Wistar rats." Carcinogenesis. 28(12):2650-2656. PMID:17724374
- [ ] Planchamp C, et al. (2007) "Function of both sinusoidal and canalicular transporters controls the concentration of organic anions within hepatocytes." Mol Pharmacol. 71(4):1089-1097. PMID:17234897
- [ ] Fouassier L, et al. (2007) "Hypoxia-induced changes in the expression of rat hepatobiliary transporter genes." Am J Physiol Gastrointest Liver Physiol. 293(1):G25-G35. PMID:17615179
- [ ] Li GX, et al. (2007) "Protective effects of hepatocellular canalicular conjugate export pump (Mrp2) on sodium arsenite-induced hepatic dysfunction in rats." Exp Toxicol Pathol. 58(6):447-453. PMID:17467962
- [ ] Mottino AD, et al. (2007) "Disruption of function and localization of tight junctional structures and Mrp2 in sustained estradiol-17beta-D-glucuronide-induced cholestasis." Am J Physiol Gastrointest Liver Physiol. 293(1):G391-G402. PMID:17463180
- [ ] Okada K, et al. (2007) "Inchinkoto, a herbal medicine, and its ingredients dually exert Mrp2/MRP2-mediated choleresis and Nrf2-mediated antioxidative action in rat livers." Am J Physiol Gastrointest Liver Physiol. 292(5):G1450-G1463. PMID:17038627
- [ ] Chiba M, et al. (2007) "Down-regulation of intestinal multidrug resistance-associated protein 2 in long-evans cinnamon rats." Drug Metab Pharmacokinet. 22(6):450-455. PMID:18159133
- [ ] Notenboom S, et al. (2006) "Increased apical insertion of the multidrug resistance protein 2 (MRP2/ABCC2) in renal proximal tubules following gentamicin exposure." J Pharmacol Exp Ther. 318(3):1194-1202. PMID:16757538
- [ ] Suzuki T, et al. (2006) "Gender-related differences in expression and function of hepatic P-glycoprotein and multidrug resistance-associated protein (Mrp2) in rats." Life Sci. 79(5):455-461. PMID:16483613
- [ ] Simon FR, et al. (2006) "Hormonal regulation of hepatic multidrug resistance-associated protein 2 (Abcc2) primarily involves the pattern of growth hormone secretion." Am J Physiol Gastrointest Liver Physiol. 290(4):G595-G608. PMID:16537972
- [ ] Bolder U, et al. (2006) "Heat stress enhances recovery of hepatocyte bile acid and organic anion transporters in endotoxemic rats by multiple mechanisms." Cell Stress Chaperones. 11(1):89-100. PMID:16572733
- [ ] de Waart DR, et al. (2006) "Multidrug resistance associated protein 2 mediates transport of prostaglandin E2." Liver Int. 26(3):362-368. PMID:16584400
- [ ] Gao Y, et al. (2006) "[Effects of MRP2-GSH cotransport system on hepatic arsenic metabolism in rats]" Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 24(5):278-280. PMID:16737587
- [ ] Sekine S, et al. (2006) "Oxidative stress and Mrp2 internalization." Free Radic Biol Med. 40(12):2166-2174. PMID:16785030
- [ ] Zamek-Gliszczynski MJ, et al. (2006) "Differential involvement of Mrp2 (Abcc2) and Bcrp (Abcg2) in biliary excretion of 4-methylumbelliferyl glucuronide and sulfate in the rat." J Pharmacol Exp Ther. 319(1):459-467. PMID:16857726
- [ ] Shibayama Y, et al. (2006) "Effect of methotrexate treatment on expression levels of multidrug resistance protein 2, breast cancer resistance protein and organic anion transporters Oat1, Oat2 and Oat3 in rats." Cancer Sci. 97(11):1260-1266. PMID:16925582
- [ ] Nishimura M, et al. (2006) "Regulation of mRNA expression of MDR1, MRP1, MRP2 and MRP3 by prototypical microsomal enzyme inducers in primary cultures of human and rat hepatocytes." Drug Metab Pharmacokinet. 21(4):297-307. PMID:16946557
- [ ] Geier A, et al. (2005) "Characterization of organic anion transporter regulation, glutathione metabolism and bile formation in the obese Zucker rat." J Hepatol. 43(6):1021-1030. PMID:16139386
- [ ] Mottino AD, et al. (2005) "Role of microtubules in estradiol-17beta-D-glucuronide-induced alteration of canalicular Mrp2 localization and activity." Am J Physiol Gastrointest Liver Physiol. 288(2):G327-G336. PMID:15374814
- [ ] Jones BR, et al. (2005) "The role of protein synthesis and degradation in the post-transcriptional regulation of rat multidrug resistance-associated protein 2 (Mrp2, Abcc2)." Mol Pharmacol. 68(3):701-710. PMID:15917434
- [ ] Hesselink DA, et al. (2005) "Cyclosporine interacts with mycophenolic acid by inhibiting the multidrug resistance-associated protein 2." Am J Transplant. 5(5):987-994. PMID:15816878
- [ ] Dietrich CG, et al. (2004) "Consequences of bile duct obstruction on intestinal expression and function of multidrug resistance-associated protein 2." Gastroenterology. 126(4):1044-1053. PMID:15057744
- [ ] Smeets PH, et al. (2004) "Contribution of multidrug resistance protein 2 (MRP2/ABCC2) to the renal excretion of p-aminohippurate (PAH) and identification of MRP4 (ABCC4) as a novel PAH transporter." J Am Soc Nephrol. 15(11):2828-2835. PMID:15504935
- [ ] Kudo A, et al. (2004) "Kupffer cells alter organic anion transport through multidrug resistance protein 2 in the post-cold ischemic rat liver." Hepatology. 39(4):1099-1109. PMID:15057914
- [ ] Chang TH, et al. (2004) "Expression of MRP2 and MRP3 during liver regeneration after 90% partial hepatectomy in rats." Transplantation. 77(1):22-27. PMID:14724430
- [ ] Reichard JF, et al. (2003) "Characterization of multidrug resistance-associated protein 2 in the hepatocellular disposition of 4-hydroxynonenal." Arch Biochem Biophys. 411(2):243-250. PMID:12623073
- [ ] Potschka H, et al. (2003) "Brain access and anticonvulsant efficacy of carbamazepine, lamotrigine, and felbamate in ABCC2/MRP2-deficient TR- rats." Epilepsia. 44(12):1479-1486. PMID:14636316
- [ ] Norimizu S, et al. (2003) "Carbon monoxide stimulates mrp2-dependent excretion of bilirubin-IXalpha into bile in the perfused rat liver." Antioxid Redox Signal. 5(4):449-456. PMID:13678533
- [ ] Geier A, et al. (2003) "Effects of proinflammatory cytokines on rat organic anion transporters during toxic liver injury and cholestasis." Hepatology. 38(2):345-354. PMID:12883478
- [ ] Potschka H, et al. (2003) "Multidrug resistance protein MRP2 contributes to blood-brain barrier function and restricts antiepileptic drug activity." J Pharmacol Exp Ther. 306(1):124-131. PMID:12663688
- [ ] Tomer G, et al. (2003) "Differential developmental regulation of rat liver canalicular membrane transporters Bsep and Mrp2." Pediatr Res. 53(2):288-294. PMID:12538788
- [ ] Tygstrup N, et al. (2002) "Messenger RNA profiles in liver injury and stress: a comparison of lethal and nonlethal rat models." Biochem Biophys Res Commun. 290(1):518-525. PMID:11779202
- [ ] Li X, et al. (2002) "Mrp2 modulates the activity of chloride channels in isolated hepatocytes." Hepatology. 36(1):65-71. PMID:12085350
- [ ] Tanaka Y, et al. (2002) "Increased renal expression of bilirubin glucuronide transporters in a rat model of obstructive jaundice." Am J Physiol Gastrointest Liver Physiol. 282(4):G656-G662. PMID:11897625
- [ ] Ito K, et al. (2001) "Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate." Am J Physiol Gastrointest Liver Physiol. 281(4):G1034-G1043. PMID:11557524
- [ ] Buchler M, et al. (1996) "cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats." J Biol Chem. 271(25):15091-15098. PMID:8662992
- [ ] Paulusma CC, et al. (1996) "Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene." Science. 271(5252):1126-1128. PMID:8599091
OBJECTIVES: We tested the effect of kidney-specific multidrug resistance-related protein (MRP2, ABCC2) deficiency on renal organic solute disposition as well as on renal protein and gene expression. Furthermore, we investigated whether a particular kidney donor ABCC2 genotype is associated with delayed graft function in patients. METHODS: A new MRP2-deficient rat strain was established. Renal cross-transplantations were performed between congenic MRP2-deficient and wild-type rats. Renal disposition of MRP2 substrates was investigated in native and transplanted rats. Proteomic analyses and transcriptional profiling were performed in rat kidney graft cortices. Ninety-eight human kidney donor-recipient pairs were genotyped for five ABCC2 polymorphisms. The relationship between delayed graft function and ABCC2 genetic variants in donors and recipients was analyzed by backward stepwise logistic regression. RESULTS: In rats, the absence of renal MRP2 reduced renal bilirubin glucuronide excretion at pathologic plasma concentrations, modified renal p-aminohippurate excretion and did not affect renal morphine-6-glucuronide excretion. Renal MRP2 deficiency led to renal cortical protein or mRNA upregulation of glutathione transferase isoenzymes, glutaredoxin 2, and heme oxygenase-1. In patients, a particular donor ABCC2 genotype was associated with an increased incidence of delayed graft function. CONCLUSION: Kidney graft-specific MRP2 deficiency has mild effects on the renal excretion of some organic solutes under experimental conditions and induces a protein and gene expression pattern indicative of activated antioxidant defense mechanisms. This suggests that MRP2 is a determinant of the redox status in tubular epithelial cells and thus of the susceptibility to renal damage under conditions of treatment with multiple drugs and increased oxygen radical formation.
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Mrp2 internalization, observed in this study, may play an important determinant of cholestasis in the post-warm ischemic livers.
2, 3-Dimercaptopropane-1-sulfonic acid (DMPS) and meso-2, 3-Dimercaptosuccinic acid (DMSA) are dithiols used to treat humans exposed to methylmercury (CH(3)Hg(+)). After treatment, significant amounts of mercury are eliminated rapidly from the kidneys and are excreted in urine. In the present study, we extended our previous studies by testing the hypothesis that MRP2 mediates the secretion of DMPS or DMSA S-conjugates of CH(3)Hg(+). To test this hypothesis, the disposition of mercury was assessed in control and Mrp2-deficient (TR(-)) rats exposed intravenously to a 5.0-mg/kg dose of CH(3)HgCl. Twenty-four and 28 h after exposure, groups of four control and four TR(-) rats were injected with saline, DMPS, or DMSA. Tissues were harvested 48 h later. Renal and hepatic contents of mercury were greater in saline-injected TR(-) rats than in controls. In contrast, the amounts of mercury excreted in urine and feces by TR(-) rats were less than those by controls. DMPS and DMSA significantly reduced the renal and hepatic content of mercury in both groups of rats, with the greatest reduction in controls. A significant increase in urinary and fecal excretion of mercury (which was greater in the controls) was also observed. Our findings in inside-out membrane vesicles prepared from hMRP2-transfected Sf9 cells show that uptake of DMPS and DMSA S-conjugates of CH(3)Hg(+) was greater in the vesicles containing hMRP2 than in control vesicles. Overall, these dispositional findings indicate that MRP2 does play a role in DMPS- and DMSA-mediated elimination of mercury from the kidney.
Multidrug resistance-associated protein 2 (MRP2) (ABCC2) is an ATP-binding cassette membrane protein located primarily on apical surface of hepatocytes that mediates transport of conjugated xenobiotics and endogenous compounds into bile. MRP2 is highly expressed in hepatocytes, and at lower levels in small intestines, stomach and kidney. Previous reports have characterized mammalian MRP2 promoters, but none have established the molecular mechanism(s) involved in liver enriched expression. This study aims to investigate the mechanism of hepatic MRP2 regulation. A 2130 bp of MRP2 promoter was cloned from PAC-1 clone P108G1-7, to identify putative liver specific/hormone responsive functional DNA binding sites. Using deletion analysis, site specific mutagenesis and co-transfection studies, liver specific expression was determined. MRP2 promoter-LUC constructs were highly expressed in liver cell lines compared to non-liver cells. The region extending from -3 to+458 bp of MRP2 promoter starting from AUG contained the potential binding sites for CAATT box enhancer binding protein (C/EBP), hepatocytes nuclear factor 1, 3 and 4 (HNF1, HNF3, and HNF4. Only HNF1 and HNF4 co-transfection with MRP2 luciferase increased expression. Site specific mutational analysis of HNF1 binding site indicated an important role for HNF1alpha. HNF4alpha induction of MRP2 was independent of HNF1 binding site. C/EBP, HNF3, and HNF6 inhibited HNF1alpha while HNF4alpha induced MRP2 luciferase expression and glucocorticoids stimulated MRP2 expression. This study emphasizes the complex regulation of MRP2 with HNF1alpha and HNF4alpha playing a central role. The coordinated regulation of xenobiotic transporters and oxidative conjugation may determine the adaptive responses to cellular detoxification processes.
The ATP-binding cassette transporter family C 2 (Abcc2) is a member of efflux transporters involved in the biliary excretion of organic anions from hepatocytes. Posttranslational regulation of Abcc2 has been implicated, although the molecular mechanism is not fully understood. In the present study, we performed yeast two-hybrid screening to identify novel protein(s) that particularly interacts with the linker region of Abcc2 located between the NH(2)-terminal nucleotide binding domain and the last membrane-spanning domain. The screening resulted in the identification of a series of small ubiquitin-like modifier (SUMO)-related enzymes and their substrates. In yeast experiments, all of these interactions were abolished by substituting the putative SUMO consensus site in the linker region (IKKE) in Abcc2 to IRKE. In vitro SUMOylation experiments confirmed that the Abcc2 linker was a substrate of Ubc9-mediated SUMOylation. It was also found that the IKKE sequence is the target of SUMOylation, since a mutant with IKKE is substituted by IRKE was not SUMOylated. Furthermore, we demonstrated for the first time that Abcc2, endogenously expressed in rat hepatoma-derived McARH7777 cells, is SUMOylated. Suppression of endogenous Ubc9 by small interfering RNA resulted in a selective 30% reduction in Abcc2 protein expression in the postnuclear supernatant, whereas subcellular localization of Abcc2 confirmed by semiquantitative immunofluorescence analysis was minimally affected. This is the first demonstration showing the regulation of ABC transporter expression by SUMOylation.
Various mechanisms can influence the intestinal absorption and oral bioavailability of drugs. The barrier effects of efflux transporters may be one of the critical factors limiting the bioavailability of certain drugs. It has been reported that multidrug resistance-associated protein 2 (Mrp2) is expressed in the mucosal membrane of the epithelium of the small intestine and secretes various drugs into the jejunum lumen. However, it is possible that total intestinal secretion of Mrp2 substrates is accounted for the contribution of Mrp2 and other transporter(s) to the intestinal secretion of Mrp2 substrates. In this study, we found that phenolsulfonphthalein and pravastatin, both Mrp2 substrates, are transported by different transport systems in the intestine. These results suggest that contribution of transporters to the drug transport may be a critical factor affecting drug disposition and drug-drug interaction. In addition to evaluating the substrate specificity of a transporter, it is important to be aware of the contribution of a transporter to drug disposition.
Bile salts may initiate or aggravate cholestasis in man. Infusion of Taurochenodeoxycholate (TCDCA) represents a model of bile salt-induced cholestasis in rat. The events leading to cholestasis are incompletely understood. The canalicular conjugate export pump Mrp2 is the major driving force for the bile salt-independent bile flow. Redistribution of Mrp2 has been suggested to cause reduction in bile flow in others models of acute cholestasis (i.e. endotoxin, phalloidin, GSH-depletion). We have studied the effects of TCDCA on the distribution of Mrp2 and P-glycoproteins with respect to changes in the actin cytoskeleton and actin associated proteins radixin and ZO-1. Bile duct cannulated rats were infused with TCDCA (0.1 and 0.4 micromol/min/100g body weight) and bile flow was measured. After 30 min livers were removed and distribution of Mrp2, P-glycoproteins, actin, actin-associated radixin and ZO1 were studied by immunofluorescence analysis. TCDCA at subcholestatic amounts (0.1 micromol/min/100 g body weight) led to distortion and dilation of the canaliculi which was apparent in actin, ZO-1, and Mrp2 fluorescence. Administration of higher amounts of TCDCA (0.4 micromol/min/100g body weight) led to a reduction of bile flow to 31 % of control bile flow. Radixin, which localized strictly to the plasmamembrane in controls, was detected in intracellular structures partially colocalizing with actin aggregates especially at the sinusoidal membranes as visualized by double-immunofluorescence staining. Mrp2 appeared in pericanalicular membrane structures in cholestatic animals whereas P-glycoproteins remained unchanged under these conditions. CONCLUSIONS: Bile salt-induced cholestasis is associated with changes of the actin cytoskeleton and actin binding protein radixin and a retrieval of the canalicular export pump Mrp2.
The drug transporter, multidrug resistance-associated protein 2 (ABCC2/Mrp2), is known to play important roles in excretion of various drugs. In the present study, we investigated whether Mrp2 is involved in the transport of micafungin, a newly developed antifungal agent. When Sprague-Dawley rats received an intravenous injection of micafungin (1 mg/kg) in combination with cyclosporine, the cyclosporine significantly delayed the disappearance of micafungin from plasma and decreased the systemic clearance and volume of distribution at steady-state of micafungin to 54% and 65% of the corresponding control values, respectively. When Sprague-Dawley rats received a constant-rate infusion of micafungin, cyclosporine significantly decreased the steady-state biliary clearance of micafungin (approximately 80%). A significant decrease in the biliary clearance of micafungin (~60%) was observed in Eisai hyperbilirubinemic rats, which have a hereditary deficiency in Mrp2. The present findings at least suggest that Mrp2 is involved mainly in the hepatobiliary excretion of micafungin in rats.
The present study was carried out in order to identify the changes in expression of multidrug resistance-associated protein (Mrp) 2 and P-glycoprotein (P-gp) in the intestine and remote organs after intestinal ischemia-reperfusion (I/R). Mrp2 expression in the jejunum and liver was decreased at 6 h after I/R. This decrease in Mrp2 expression was associated with an increase in the serum level of IL-6. These results suggest that the decreased Mrp2 expression after intestinal I/R was regulated by IL-6. The expression level of mdr1a in the ileum, which encodes P-gp, was decreased at 6 and 24 h after I/R, and the expression level of mdr1b, also encodes P-gp, was not altered at any time. P-gp protein expression in the ileum was decreased at 6 h after I/R. In the liver, mdr1a expression was decreased at 6 h after I/R, but mdr1b expression was increased at 6 h after I/R. P-gp protein was not altered at any time. In the kidney, mdr1a expression was decreased at 24 h after I/R, but mdr1b expression was not altered at any time. P-gp protein expression in the kidney was decreased at 24 h after I/R, as was mdr1a expression. These results suggest that P-gp expression after intestinal I/R differs in each organ. This is the first report to provide evidence that expression levels of transporters in remote organs are altered intestinal after I/R.
Intestinal ATP binding cassette (ABC) transporters may affect the bioavailability and effectiveness of orally administered drugs. Available studies on regional expression of intestinal efflux transporters were done with selected intestinal segments only and inconsistent with regard to the variability of transporter expression and the course of expression along the intestine. For an evaluation of the consistency between mRNA and protein expression, relative expression levels of P-glycoprotein (Pgp; ABCB1), breast cancer resistance protein (Bcrp; ABCG2), and multidrug resistance-associated protein (Mrp) 2 (ABCC2) were determined using quantitative real-time-polymerase chain reaction and Western blot in rat intestinal segments from duodenum, jejunum, ileum, and colon. In addition, the protein expression of Pgp, Bcrp, and Mrp2 from the entire rat intestine was studied by a complete 3-cm segmentation to evaluate the predictive power of expression analyses from selected intestinal segments. Pgp showed an increase from proximal to distal regions, Bcrp showed an arcuate pattern with highest expression toward the end of small intestine, and Mrp2 decreased along the intestinal axis from proximal to distal parts. No gender specific differences could be observed. Regarding the concordance of mRNA and protein expression, Pgp and Bcrp mRNA samples allow good estimations about the corresponding protein expression (for Pgp limited to the mdr1a isoform), but for Mrp2, pronounced deviation could be observed. All transporters showed considerable intra- and interindividual variability, especially at the protein level, making it problematic to take transporter expressions of small sections exemplary for general assumptions on intestinal abundances.
Hepatic ischemia-reperfusion (IR) injury during liver transplantation can lead to cholestasis and remote organ dysfunction. Multidrug resistance-associated proteins (Mrps) are efflux transporters known to transport a diverse set of substrates, such as amphipathic chemicals, organic anions, and endogenous molecules. The purpose of this study was to determine the effect of hepatic IR injury on the expression of Mrps in rat liver and kidney. Male Sprague-Dawley rats were subjected to 60 min of partial hepatic ischemia. At various times after reperfusion (0, 3, 6, 24, and 48 h), the ischemic lobes were harvested as well as kidneys. RNA and protein expression of Mrps in livers and kidneys were determined by the branched DNA method, Western blot analysis, and tissue immunofluorescence. Mrp2 mRNA and protein expression in livers decreased after IR. Conversely, Mrp2 mRNA and protein expression in kidneys increased after IR. Mrp3 mRNA expression, and Mrp4 mRNA and protein expression in kidneys transiently increased after IR. The intensity of immunofluorescent staining of Mrp2 corresponded to changes in Mrp2 expression in livers and kidneys after IR as detected by Western blot analysis and was localized to the apical membrane domain in both tissues. These results demonstrate that after hepatic IR, downregulation of hepatic Mrp2 and upregulation of renal Mrp2 occur. These decreases in hepatic Mrp2 may contribute to cholestasis, yet increases in kidney may protect from oxidative stress and/or inflammation after hepatic IR.
BACKGROUND AND AIM: The present study was aimed to evaluate the hepatic zonation of multidrug resistance-associated protein 2 (mrp2), an important drug transporter, and its potential changes during the induction of its expression by known inducer, dexamethasone (DEX). METHODS: The hepatic expression of mrp2 was studied by immunohistochemistry with consequent quantification by measurement of integral optical densities of mrp2 staining in the periportal and perivenous areas of the liver acinus in control and DEX-pretreated rats (1 mg/kg daily per os for 4 days). Overall changes in mrp2 expression and function produced by DEX were monitored using Western blotting and an in vivo clearance study of endogenous-conjugated bilirubin, a mrp2 substrate. RESULTS: In the control animals, a quantitative image analysis revealed the primary periportal localization of mrp2 within the liver acinus with the expression of mrp2 being 16.7-fold of that in the perivenous area. After DEX pretreatment, the expression of mrp2 increased, especially in the perivenous hepatocytes. The overall expression of mrp2 increased 3.2-fold in comparison with the control group. This observation was confirmed by Western blotting, which showed a 1.3-fold increase in the mrp2 protein after DEX pretreatment. The functional consequences of the induced mrp2 protein in the livers of the DEX-pretreated rats were demonstrated by the increased biliary excretion of conjugated bilirubin. CONCLUSION: In conclusion, these results indicate the zonation of mrp2 protein expression primarily to periportal hepatocytes. The induction by DEX produced spatially disproportional changes with an increase in the mrp2 protein being most prominent in the perivenous hepatocytes.
Chronic ethanol consumption elicits detrimental changes of liver metabolism. By employing a 12-week-long feeding regimen, we investigated the effects of chronic ethanol consumption on the expression and localization of bile salt export pump (Bsep), a major canalicular exporter of bile salts, and multidrug resistance protein 2 (Mrp2), a canalicular organic anion transporter, in the rat liver. RT-PCR, confocal immunofluorescence microscopy, immunoblotting, and quantitative colocalization analysis were used to examine their gene and protein expression, and changes in the distribution of antigenic sites. Bsep mRNA was upregulated, while Mrp2 mRNA responded by downregulation. In agreement with mRNA, the expression of Bsep protein increased, while the expression of Mrp2 protein responded with a decrease, suggesting that the expression of both of them is transcriptionally regulated. Confocal immunofluorescence microscopy showed disruption of the colocalization of Bsep and Mrp2 proteins at the hepatocyte canalicular membrane and their relocation intracellularly. Quantitative colocalization analysis of Bsep and Mrp2 proteins revealed a steady decrease in the degree of colocalization and Mrp2 expression, indicating that although the properties of both transporters are affected, Mrp2 is altered more. These findings provide evidence that ethanol alters Bsep and Mrp2 canalicular transporters in the rat liver, at both the mRNA and protein levels. Mrp2 shows deeper involvement. Eight weeks appears to be a critical time point in this process.
The role of uptake and efflux transport proteins in the tissue distribution of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolites is largely unknown. Carbonyl reduction of NNK results in formation of the carcinogenic 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which in rats is glucuronidated to the non-toxic NNAL-O-glucuronide. Previous in vitro studies showed that NNAL-O-glucuronide is a substrate for the human ATP-binding cassette transport proteins multidrug resistance protein (MRP)1 (ABCC1) and MRP2 (ABCC2). To investigate the influence of Mrp2 deficiency on NNK biotransformation and biliary excretion, [(3)H]NNK was administered intravenously to bile duct-cannulated wild-type (WT) and Mrp2-deficient (TR(-)) Wistar rats; plasma, bile and urine samples were collected for 5 h and analyzed by high-pressure liquid chromatography with radiochemical detection. The total radioactivity recovered in WT and TR(-) bile was 12 and 7% of the dose, respectively. NNAL-O-glucuronide accounted for 87% of the radioactivity in WT bile but was not detected in TR(-) bile. Urinary recovery of 1-(3-pyridyl)-1-butanol-4-carboxylic acid (hydroxy acid), NNAL-O-glucuronide and NNAL-N-oxide from 2-5 h was greater in TR(-) compared with WT rats. NNK plasma clearance was significantly higher in TR(-) (115 +/- 23 ml/min/kg) compared with WT (48 +/- 13 ml/min/kg) rats. A higher concentration and/or earlier appearance of hydroxy and 1-(3-pyridyl)-1-butanone-4-carboxylic acids, NNAL-N-oxide and NNK-N-oxide, and decreased NNK and NNAL concentrations in TR(-) plasma suggested increased cytochrome P450 biotransformation in TR(-) rats. The total recovery of hydroxy acid in bile and urine was significantly higher in TR(-) compared with WT rats. Thus, Mrp2 is responsible for the biliary excretion of NNAL-O-glucuronide and Mrp2 deficiency results in increased formation of carcinogenic NNK metabolites.
We hypothesized that the function of both sinusoidal and canalicular transporters importantly controls the concentrations of organic anions within normal hepatocytes. Consequently, we investigated how acute transport regulation of the sinusoidal organic anion transporting polypeptides (Oatps) and the canalicular multidrug resistance associated protein 2 (Mrp(2)) determines the hepatic concentrations of the organic anion gadolinium benzyloxypropionictetraacetate (BOPTA) in rat livers. Livers were perfused with labeled BOPTA in different experimental settings that modify the function of Oatps and Mrp(2) through the protein kinase C (PKC) pathway. Intrahepatic concentrations were continuously measured with a gamma probe placed above rat livers. Labeled BOPTA was also measured in perfusate and bile. We showed that when the function of Oatps and Mrp(2) is modified in such a way that BOPTA entry and exit are similarly decreased, concentrations of organic anions within hepatocytes remain unaltered. When exit through Mrp(2) is abolished, hepatic concentrations are high if entry through Oatps is only slightly decreased (livers without Mrp(2) expression) or low if BOPTA uptake is more importantly decreased (livers perfused with a PKC activator). These results highlight that the function of both sinusoidal and canalicular transporters is important to determine the concentration of organic anions within hepatocytes.
Cholestatic disorders may arise from liver ischemia (e.g., in liver transplantation) through various mechanisms. We have examined the potential of hypoxia to induce changes in the expression of hepatobiliary transporter genes. In a model of arterial liver ischemia subsequent to complete arterial deprivation of the rat liver, the mRNA levels of VEGF, a hypoxia-inducible gene, were increased fivefold after 24 h. The pattern of VEGF-induced expression and ultrastructural changes, including swelling of the endoplasmic reticulum, indicated that hypoxia affected primarily cholangiocytes, but also hepatocytes, predominantly in the periportal area. Serum and bile analyses demonstrated liver dysfunction of cholestatic type with reduced bile acid biliary excretion. Fluorescence-labeled ursodeoxycholic acid used as a tracer displayed no regurgitation, eliminating bile leakage as a significant mechanism of cholestasis in this model. In liver tissue, a marked reduction in the mRNA levels of Na(+)-taurocholate-cotransporting polypeptide (Ntcp), bile salt export protein (Bsep), and multidrug resistance-associated protein 2 (Mrp2) and an increase in those of Cftr were detected before bile duct proliferation occurred. In cultured hepatocytes, a nontoxic hypoxic treatment caused a decrease in the mRNA and protein expression of Ntcp, Bsep, and Mrp2 and in the mRNA levels of nuclear factors involved in the transactivation of these genes, i.e., HNF4alpha, RXRalpha, and FXR. In bile duct preparations, hypoxic treatment elicited an increase in Cftr transcripts, along with a rise in cAMP, a major regulator of Cftr expression and function. In conclusion, hypoxia triggers a downregulation of hepatocellular transporters, which may contribute to cholestasis, whereas Cftr, which drives secretion in cholangiocytes, is upregulated.
Arsenic is a double-edged sword to human health. The excretion of various organic anions into bile is mediated by an adenosine triphosphate-dependent conjugate export pump, which has been identified as the canalicular isoform of the multidrug resistance protein 2 (Mrp2). It has been proved that Mrp2 can transport arsenite in vitro, but its effects in vivo are not clear. The aim of this study was to investigate whether Mrp2 plays a role in exportation of arsenic in vivo and its protective effects on liver function. Mrp2 protein level in rat liver was determined by Western blot analysis. Total arsenic concentrations in whole blood and bile were measured using hydride generation atomic absorption spectrometry. Alanine aminotransferase (ALT) activity, aspartate aminotransferase activity (AST), glutathione peroxidase (GSH-PX) activity, malon dialdehyde (MDA) and total bilirubin were measured by biochemical assays. The morphological changes were observed by electron microscopy. Total arsenic levels in blood and bile of arsenite-treated rats were significantly higher than those of control rats (P<0.05) at all three different time points. The overexpression of Mrp2 was 36.61%, 32.36% and 12.73% at 2, 4 and 6 weeks, respectively (percentage of controls, P<0.05), which was significantly higher than controls. A positive correlation between Mrp2 expression level and total arsenic concentration in bile indicated that Mrp2 accelerated the transport of arsenic. Electron microscopy showed that microvilli of bile canaliculi became swollen and sparse. ALT and AST activities in serum were markedly raised at 6 weeks. MDA level in serum increased (P<0.05) and GSH-PX activity in serum decreased except for 2 weeks. Damage of liver function became worse following decreased expression of Mrp2. In conclusion, overexpression of Mrp2 may explain increased biliary excretion of arsenic and it may protect liver function.
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.
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.
Wilson's disease is an inherited, autosomal recessive disorder of copper accumulation and toxicity. Lifelong chelation therapy is essential in all Wilson's disease patients. Intestinal absorption of some compounds is limited partly because they are preferentially transported in the secretory direction. Several ATP-binding cassette (ABC) transporters are expressed in the apical membrane of the small intestine and secrete various drugs into the lumen. In this study, we investigated the characteristics of the intestinal efflux ABC transporters in LEC rats. We found that the expression of multidrug resistance-associated protein 2 (Mrp2) in the jejunum of Long-Evans Cinnamon (LEC) rats, an animal model for Wilson's disease, is decreased.
Multidrug resistance protein (MRP) 2 (MRP2; ABCC2), an organic anion transporter apically expressed in liver, kidney, and intestine, plays an important protective role through facilitating the efflux of potentially toxic compounds. We hypothesized that upon a toxic insult, MRP2 is up-regulated in mammalian kidney, thereby protecting the tissue from damage. We studied the effects of the nephrotoxicant gentamicin on the functional expression of MRP2 in transfected Madin-Darby canine kidney type II (MDCKII) cells and rat kidney. Transport of glutathionemethyl fluorescein by cells or calcein by isolated perfused rat kidney was measured to monitor MRP2 activity. MDCKII cells were exposed to gentamicin (0-1000 microM) for either 1 h, 24 h, or for 1 h followed by 24-h recovery. No effect was observed on MRP2 after 1-h exposure. After 24-h gentamicin exposure or after a 24-h recovery period following 1-h exposure, an increase in MRP2-mediated transport was seen. This up-regulation was accompanied by a 2-fold increase in MRP2 protein expression in the apical membrane, whereas the expression in total cell lysates remained unchanged. In perfused kidneys of rats exposed to gentamicin (100 mg/kg) for seven consecutive days, an increase in Mrp2 function and expression was found, which was prevented by addition of a dual endothelin-receptor antagonist, bosentan. We conclude that an increased shuttling of the transporter to the apical membrane takes place in response to gentamicin exposure, which is triggered by endothelin. Up-regulation of MRP2 in the kidney may be interpreted as part of a protective mechanism.
To clarify whether gender-related differences exist in the expression and function of hepatic P-glycoprotein- and/or multidrug resistance-associated protein (Mrp2), we measured the hepatobiliary excretion of doxorubicin and their protein levels in male and female Sprague-Dawley rats. When rats received a single intravenous injection of doxorubicin (5 mg/kg), a delay in the disappearance of doxorubicin from plasma was observed in male rats. When rats received a constant-rate infusion of doxorubicin, no significant gender-related differences in the apparent biliary clearance of doxorubicin based on the steady state plasma concentrations were observed between male and female rats. However, the net biliary clearance of doxorubicin based on the liver concentration, which represents the actual function of P-glycoprotein and/or Mrp2, was higher in female rats than in male rats. These results suggest that the actual function of the hepatobiliary transport of doxorubicin is greater in female than in male rats. Western blot analysis revealed that the expression of P-glycoprotein and Mrp2 in the liver of female rats was significantly higher than in male rats, similar to results of hepatobiliary excretion experiments. The expression of hepatic cytochrome P450 (CYP) 2B1, which is involved in the metabolism of doxorubicin, was significantly higher in male than in female rats. By pretreatment with testosterone (10 mg/day for 7 days), the actual biliary clearance of doxorubicin in female rats was nearly that of male rats. The protein levels of P-glycoprotein and Mrp2 in female rats were also lowered by treatment with testosterone so as to be nearer those in male rats. These results suggest that gender-related differences exist in P-glycoprotein- and Mrp2-mediated hepatobiliary transport and that these two transporters may be regulated by sex hormones.
Biliary excretion is the rate-limiting step in transfer of bilirubin, other organic anions, and xenobiotics across the liver. Multidrug resistance-associated protein 2 (Mrp2, Abcc2) is the major transporter for conjugated endo- and xenobiotic-conjugated compounds into bile. Hormones regulate bilirubin and xenobiotic secretion into bile, which have dimorphic differences. Therefore, we examined the possible role of sex steroids and growth hormone in the regulation of Mrp2. In approximately 8-wk-old rats, mRNA, transcriptional activity, and hepatic content of Mrp2 were selectively increased fourfold (P < 0.001) in females compared with males. In males, estrogens increased and testosterone decreased Mrp2 mRNA and protein, whereas no significant effect was measured in females, suggesting either a direct effect on the liver or an alteration in growth hormone secretory pattern. After hypophysectomy, Mrp2 mRNA was markedly reduced and the effects of estrogens and testosterone on Mrp2 were prevented, supporting the role of pituitary hormones in controlling Mrp2 expression. Mrp2 increased following growth hormone infusion in males. Mrp2 mRNA was decreased in growth hormone-deficient "Little" mice. Growth hormone infusions in hypophysectomized rats partially restored Mrp2 levels, whereas thyroxine addition returned Mrp2 mRNA and protein to basal levels. Morphology as well as biochemical measurements demonstrated that Mrp2 was localized to the bile canaliculus in equal density in both genders, whereas hormone replacements increased Mrp2 in hypophysectomized animals. In cultured hepatocytes, thyroxine did not have an effect, but growth hormone alone and combined with thyroxine increased Mrp2 mRNA levels. In conclusion, Mrp2 levels are regulated by the combination of thyroxine and different growth hormone secretory patterns.
Heat stress (HS) reduces the many sequelae of lipopolysaccharide (LPS)-induced endotoxemia. Without HS, endotoxins have been shown to induce a transcriptional down-regulation of hepatocyte transport proteins for bile acids and organic anions. We performed experiments in isolated perfused rat livers at various times after LPS administration with and without HS pretreatment to determine whether HS would correct deficient transport of bromosulfophthalein (BSP). Possible mechanisms involved were investigated in livers from intact animals. In isolated perfused livers, LPS injection reduced BSP excretion to 48% compared with saline-injected controls (P < 0.01). When HS was applied 2 hours prior to LPS, BSP excretion increased to 74% of controls (P < 0.05 vs LPS and controls). Expression of the basolateral (Oatp1a1) and canalicular (Mrp2) organic anion transporter involved in the transport of BSP recovered more rapidly when HS preceded LPS application. Recovery of mRNA levels of these transporters occurred also earlier. Coimmunoprecipitation experiments and immunoelectron microscopy using a double immunogold labeling of heat shock protein 70 (HSP70) and various hepatocyte transporters suggested colocalization with HSP70 for the canalicular bile acid transporter (Bsep) in the subcanalicular space. In contrast, no colocalization was shown for Ntcp and anion transporters. In conclusion, we could show that HS enhances recovery of organic anion transporters and bile acid transporters following endotoxemia. Faster recovery of mRNA seems to be a key mechanism for anion transporters, whereas physical interaction with HSP70 plays a role in preservation of bile acid transporters. This interaction of HSP70 and canalicular transporters occurs only in pericanalicular vesicles but not when the protein is integrated into the plasma membrane.
BACKGROUND/AIM: Inactivation of prostaglandin E(2) (PGE(2)) in the liver is a rapid process and occurs mainly through beta-oxidation in the peroxisome of the hepatocyte. Biliary excretion of PGE(2) is also a means of elimination from the liver. We investigated the role of multidrug resistance-associated protein 2 (MRP2) in the transport of PGE(2). METHODS: Biliary PGE(2) elimination was measured in liver perfusions in Wistar and MRP2-deficient TR(-) rats. Furthermore, transport experiments were performed in membrane vesicles from human MRP2-infected Spodoptera frugiperda 21 (Sf21) insect cells. RESULTS: The liver perfusions showed a 3.5 times higher percentage of undegraded [(3)H]PGE(2) in bile of Wistar rats in comparison with MRP2 deficient (TR(-)) rats (3.6% vs. 1.1%, respectively; P<0.05). MRP2-mediated transport of the model substrate [(3)H]DNP-SG was inhibited by PGE(2). Half maximal inhibition was achieved at a concentration of approximately 15 microM PGE(2). In addition, [(3)H]PGE(2) uptake in these vesicles was detected, and determined to be ATP dependent. CONCLUSION: MRP2 mediates the transport of PGE(2) and its breakdown products. The biliary excretion of PGE(2) via MRP2 may contribute to rapid elimination of the prostaglandin but might also serve to relay prostaglandin signalling to the biliary tree.
OBJECTIVE: To investigate the role of multidrug resistant protein 2 (MRP2) and glutathione (GSH) cotransport system in hepatic arsenic metabolism in rats. METHODS: Thirty healthy Wistar rats were divided randomizedly into five groups. The first group was the control group and the rats in this group were administered with normal saline. In the second, third and fourth group the rats were administered with 4, 10 and 20 mg As(+)3/kg BW of sodium arsenite respectively every other day for two weeks. The fifth group was the benzene-soluble organics (BSO) intervention group and in this group the rats were administered with 2 mmol/kg BW BSO intraperitoneally every day three days before the end of the experiment. The other treatment was the same as in other groups. All rats were sacrificed two weeks after the treatments. Arsenic contents in bile, liver and blood were detected by atomic absorption spectroscopy (AAS), and the expression of MRP2 in the membrane of hepatocyte was determined by Western-blot analysis. RESULTS: The level of total arsenic (including organic arsenic and inorganic arsenic) in bile, liver and blood in all three different dose groups was higher than those in the control groups (P < 0.05). Arsenic levels of bile and liver were increased with intragastric arsenic dose. Blood arsenic levels were not significantly different in three different dose groups. Expression of hepatic MRP2 was increased with intragastric arsenic concentration. A positive correlation between biliary arsenic concentration and MRP2 levels was found in liver (r = 0.986, P < 0.05). For the rats pretreated with BSO, the biliary arsenic was significantly higher than that in the control group but lower than that in the high dose group; the liver and blood arsenic was higher than that in the control group and in the high dose group. Expression of MRP2 pretreated with BSO was decreased. CONCLUSION: Sodium arsenite can induce expression of MRP2 and the up-regulation of MRP2 may play an important role in the bile secretion of arsenite and its metabolites. The function of MRP2 for transportation of arsenic and its metabolites is associated with the intracellular GSH level. BSO inhibits the synthesis of GSH, which weakens the function of the MRP2-GSH cotransport system and makes the liver arsenic increased.
Oxidative stress in the liver is sometimes accompanied by cholestasis. We have described the internalization of multidrug resistance-associated protein 2/ATP-binding cassette transporter family 2 (Mrp2/Abcc2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress in rat liver. However, the signaling pathway and regulatory molecules have not been investigated. In the present study, we investigated the mechanism of EA-induced Mrp2 internalization using isolated rat hepatocyte couplets (IRCHs). The Mrp2 index, defined as the ratio of Mrp2-positive canalicular membrane staining in IRCHs per number of cell nuclei, was significantly reduced by treatment with EA. This reduction was abolished by a nonspecific protein kinase C (PKC) inhibitor Go6850, a Ca(2+) chelator, EGTA, but not by a protein kinase A (PKA)-selective inhibitor, a Ca(2+)-dependent conventional PKC (cPKC) inhibitor Go6976, or a protein kinase G (PKG) inhibitor (1 microM). Moreover, an increase in the intracellular Ca(2+) level and NO release into medium were observed shortly after the EA treatment. Both of these increases, as well as Mrp2 internalization, were completely blocked by EGTA. In conclusion, EA produced a reduction in GSH, Ca(2+) elevation, NO production, and nPKC activation in a sequential manner, finally leading to Mrp2 internalization.
The hepatic excretion of hydrophilic conjugates, end products of phase II metabolism, is not completely understood. In the present studies, transport mechanism(s) responsible for the biliary excretion of 4-methylumbelliferyl glucuronide (4MUG) and 4-methylumbelliferyl sulfate (4MUS) were studied. Isolated perfused livers (IPLs) from Mrp2-deficient (TR(-)) Wistar rats were used to examine the role of Mrp2 in the biliary excretion of 4MUG and 4MUS. After a 30-micromol dose of 4-methylumbelliferone, cumulative biliary excretion of 4MUG was extensive in wild-type rat IPLs (25 +/- 3 micromol) but was negligible in TR(-) livers (0.4 +/- 0.1 micromol); coadministration of the Bcrp and P-glycoprotein inhibitor GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-di hydro-5-methoxy-9-oxo-4-acridine carboxamide] had no effect on 4MUG biliary excretion in wild-type rat IPLs. In contrast, biliary excretion of 4MUS was partially maintained in Mrp2-deficient rat IPLs. Recovery of 4MUS in bile was approximately 50 to 60% lower in both control TR(-) (149 +/- 8 nmol) and wild-type IPLs with GF120918 coadministration (176 +/- 30 nmol) relative to wild-type control livers (378 +/- 37 nmol) and was nearly abolished in TR(-) IPLs in the presence of GF120918 (13 +/- 8 nmol). These changes were the result of decreased rate constants governing 4MUG and 4MUS biliary excretion. In vitro assays and perfused livers from Bcrp and P-glycoprotein gene-knockout mice indicated that 4MUS did not interact with P-glycoprotein but was transported by Bcrp in a GF120918-sensitive manner. In the rat liver, Mrp2 mediates the biliary excretion of 4MUG, whereas both Mrp2 and Bcrp contribute almost equally to the transport of 4MUS into bile.
The ATP binding cassette (ABC) transporters, multidrug resistance protein 2 (Mrp2; Abcc2) and breast cancer resistance protein (Bcrp; Abcg2), and organic anion transporters (Oats) mediate excretion of methotrexate (MTX) and many other drugs. However, it is not known whether MTX treatment leads to any changes in the expression of these transporters. We examined the effect of MTX treatment on expression of Mrp2, Bcrp and Oats in rats. MTX was single injected intraperitoneally at doses of 10, 50 and 150 mg/kg, and then Western blot analysis was performed. The levels of Mrp2, Oat1 and Oat2 on day 1 after the treatment showed no significant change. Four days after injection of 150 mg/kg MTX, the Mrp2 levels in the liver and ileum, but not in the kidney, were markedly down-regulated to 20 +/- 3.6% and 8.9 +/- 3.8% (mean +/- SEM) of controls, respectively. Renal Oat1 and Oat3 were also down-regulated to 56.4 +/- 4.3% (Oat1) and 54.3 +/- 5.5% (Oat3) of controls. These effects of MTX were almost recovered by leucovorin which rescues normal cells from MTX toxicity. MTX treatment also decreased mRNA levels of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) to 65.5 +/- 17.9% and 59.6 +/- 14.5% of controls in the liver, respectively. MTX treatment has no apparent effect on expression levels of Bcrp, cytochrome P450 2B6 and 3A1. In conclusion, these data indicate that MTX treatment down-regulates expression levels of Mrp2, Oat1 and Oat3, and its effects are recovered by leucovorin.
The mRNA induction of various transporters by rifampicin (Rif), dexamethasone (Dex) and omeprazole (Ome) was investigated in primary cultures of cryopreserved human and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. In primary cultures of human hepatocytes, mRNA levels of MDR and MRP1 were increased by about 1.5 fold and 1.3 fold, respectively, by exposure to Rif at 2 to 50 microM as compared with 0.1% DMSO-treated controls. MRP2 mRNA levels in the same human hepatocytes were significantly increased by 1.2 to 1.8 fold by exposure to Rif at 50 microM as compared with controls. In primary cultures of rat hepatocytes, Mdr1a and Mdr1b mRNA levels were not increased or only slightly increased at 24 hr by exposure to any of the inducers at 2, 10 or 50 microM. Mrp2 mRNA levels in the same rat hepatocytes were significantly increased by 7 to 45 fold by exposure to Dex at 2 microM as compared with controls. Based on the species differences observed in the present study, primary cultures of cryopreserved hepatocytes from both the human and rat should be useful in preclinical drug development for evaluating candidate drugs for transporter induction.
BACKGROUND/AIMS: Alterations in hepatobiliary transporters may render fatty livers more vulnerable against various toxic insults. METHODS: We therefore studied expression and function of key organic anion transporters and their transactivators in 8-week-old obese Zucker rats, an established model for non-alcoholic fatty liver disease. RESULTS: Compared to their heterozygous littermates, obese animals showed a significant reduction in canalicular bile salt secretion, which was paralleled by significantly diminished Oatp2 mRNA and protein levels together with reduced nuclear HNF3beta, while expression of bile salt export pump, organic anion transporter (Oatp) 1 and multidrug resistance-associated protein (Mrp) 4 were unchanged. Impaired bile salt-independent bile flow in obese rats was associated with a 50% reduction of biliary secretion of the Mrp 2 model-substrates glutathione disulfide and S-(2,4-dinitrophenyl)glutathione. In line Mrp2 protein expression was reduced by 50% in obese rats. CONCLUSIONS: Oatp2 and Mrp2 expression is decreased in fatty liver and may impair metabolism and biliary secretion of numerous xenobiotics. Reduction of bile salt secretion and absence of biliary GSH excretion may contribute to impaired bile flow and posthepatic disorders associated with biliary GSH depletion.
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.
Multidrug resistance-associated protein 2 (Mrp2, Abcc2), an organic anion transporter present in the apical membrane of hepatocytes, renal epithelial cells, and enterocytes, is postulated to undergo post-transcriptional regulation. We hypothesized that Mrp2 protein undergoes altered rates of protein synthesis or degradation consistent with different Mrp2 protein expression. We analyzed Mrp2 synthesis, expression, and degradation in control female, 19- and 20-day pregnant, and pregnenolone-16alpha-carbonitrile (PCN)-treated rats using in vivo metabolic-labeling studies with [35S]cysteine/methionine or [14C]NaHCO3, polysomal distribution analyses and ribonuclease protection assays (RPA). Mrp2 protein was significantly increased in rats treated with PCN for 2 days but significantly decreased in 19-day pregnant rats relative to controls; no significant differences were observed in Mrp2 mRNA expression among these groups. The measured half-lives of 14C-labeled Mrp2 in control, pregnant, and PCN-treated rats were 27, 36, and 22 h, respectively, and were not significantly different. The rate of incorporation of 35S into Mrp2 was highest in PCN-treated rats. Polysomal distribution analysis of Mrp2 mRNA was consistent with increased Mrp2 protein synthesis after PCN treatment. The major transcription-initiation site for rat liver determined by RPA was -98 nucleotides (nt), with other start sites observed at -213, -163, -132, and -71 nt; use of transcription sites did not differ among the groups. Differences in the degradation of Mrp2 protein cannot explain the post-transcriptional regulation of Mrp2 in control, pregnant, and PCN-treated rats. Rather, the observed difference in protein synthesis suggests an intrinsic role for the translational regulation of rat Mrp2 protein.
In mycophenolate mofetil (MMF)-treated organ transplant recipients, lower mycophenolic acid (MPA) plasma concentrations have been found in cyclosporine (CsA) compared with tacrolimus (Tac)-based immunosuppressive regimens. We previously demonstrated that CsA decreases exposure to MPA and increases exposure to its metabolite MPA-glucuronide (MPAG), possibly by interfering with the biliary excretion of MPAG. To elucidate the role of the multidrug resistance-associated protein (Mrp)-2 in the interaction between MMF and CsA, we treated three groups of 10 Mrp2-deficient rats (TR- rat) for 6 days with either vehicle, CsA (8 mg/kg) or Tac (4 mg/kg) by oral gavage. Hereafter, co-administration with MMF (20 mg/kg) was started in all groups and continued through day 14. The 24-h MPA/MPAG area under the concentration-time curve (AUC) was determined after single (day 7) and multiple MMF doses (day 14). On both study days, there were no significant differences in the mean MPA and MPAG AUC between CsA and Tac-treated animals. We conclude that the pharmacokinetics of MMF are comparable in Mrp2-deficient rats receiving either CsA or Tac as co-medication. This finding suggests that CsA-mediated inhibition of the biliary excretion of MPAG by the Mrp2 transporter is the mechanism responsible for the interaction between CsA and MMF.
BACKGROUND & AIMS: Multidrug resistance-associated protein 2 (MRP2), a transporter of organic anions in hepatocytes, renal epithelial cells, and enterocytes, is differentially regulated in liver and kidney during cholestasis, but little is known about its regulation in the intestine. METHODS: We investigated duodenal protein expression of MRP2 in male Sprague-Dawley rats with bile duct ligation (BDL) or biliary diversion as well as in 20 cholestatic patients with biliary obstruction. RESULTS: In biliary obstruction, but not biliary depletion, intestinal Mrp2 protein mass was reduced to 9.3% +/- 5.5% of controls and mRNA to 40.5% +/- 20.8% of controls after 7 days. Binding of RXR alpha:RAR alpha heterodimers to the Mrp2 promoter element was significantly reduced in BDL rats. Cytokine blockade identified IL-1 beta as the responsible inducer of Mrp2 down-regulation. In humans with obstructive cholestasis, intestinal MRP2 protein expression was reduced to 27.3% +/- 20.3% of control patients; this reduction correlated with the duration of cholestasis and was reversible after reconstitution of bile flow by stenting of the common bile duct. However, no significant differences in MRP2 mRNA levels were detected by RT-PCR in humans. Intestinal protein expression of P-glycoprotein, breast cancer resistance protein (BCRP), and MRP3 was unchanged. In BDL rats, oral bioavailability of the Mrp2 substrate and food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was elevated 2.5 times compared with sham-operated rats. CONCLUSIONS: Cholestasis promotes down-regulation of MRP2 expression in the duodenum of rats and humans. Selective down-regulation during cholestasis might be the consequence of species-specific transcriptional and posttranscriptional mechanisms and contributes to higher bioavailability of a food-derived carcinogen.
p-Aminohippurate (PAH) is the classical substrate used in the characterization of organic anion transport in renal proximal tubular cells. Although basolateral transporters for PAH uptake from blood into the cell have been well characterized, there is still little knowledge on the apical urinary efflux transporters. The multidrug resistance protein 2 (MRP2/ABCC2) is localized to the apical membrane and mediates ATP-dependent PAH transport, but its contribution to urinary PAH excretion is not known. In this report, we show that renal excretion of PAH in isolated perfused kidneys from wild-type and Mrp2-deficient (TR(-)) rats is not significantly different. Uptake of [(14)C]PAH in membrane vesicles expressing two different MRP2 clones isolated from Sf9 and MDCKII cells exhibited a low affinity for PAH (Sf9, 5 +/- 2 mM; MDCKII, 2.1 +/- 0.6 mM). Human MRP4 (ABCC4), which has recently been localized to the apical membrane, expressed in Sf9 cells had a much higher affinity for PAH (K(m) = 160 +/- 50 microM). Various inhibitors of MRP2-mediated PAH transport also inhibited MRP4. Probenecid stimulated MRP2 at low concentrations but had no effect on MRP4; but at high probenecid concentrations, both MRP2 and MRP4 were inhibited. Sulfinpyrazone only stimulated MRP2, but inhibited MRP4. Real-time PCR and Western blot analysis showed that renal cortical expression of MRP4 is approximately fivefold higher as compared with MRP2. MRP4 is a novel PAH transporter that has higher affinity for PAH and is expressed more highly in kidney than MRP2, and may therefore be more important in renal PAH excretion.
Although Kupffer cells (KCs) may play a crucial role in post-cold ischemic hepatocellular injury, their role in nonnecrotic graft dysfunction remains unknown. This study examined reveal the role of KC in post-cold ischemic liver grafts. Rat livers treated with or without liposome-encapsulated dichloromethylene diphosphonate, a KC-depleting reagent, were stored in University of Wisconsin (UW) solution at 4 degrees C for 8 to 24 hours and reperfused while monitoring biliary output and constituents. The ability of hepatocytes to excrete bile was assessed through laser-confocal microfluorography in situ. Cold ischemia-reperfused grafts decreased their bile output significantly at 8 hours without any notable cell injury. This event coincided with impaired excretion of glutathione and bilirubin-IXalpha (BR-IXalpha), suggesting delayed transport of these organic anions. We examined whether intracellular relocalization of multidrug resistance protein-2 (Mrp2) occurred. Kinetic analyses for biliary excretion of carboxyfluorescein, a fluoroprobe excreted through this transporter, revealed significant delay of dye excretion from hepatocytes into bile canaliculi. The KC-depleting treatment significantly attenuated this decline in biliary anion transport mediated through Mrp2 in the 8-hour cold ischemic grafts via redistribution of Mrp2 from the cytoplasm to the canalicular membrane. Furthermore, thromboxane A(2) (TXA(2)) synthase in KC appeared involved as blocking this enzyme improved 5-carboxyfluorescein excretion while cytoplasmic internalization of Mrp2 disappeared in the KC-depleting grafts. In conclusion, KC activation is an important determinant of nonnecrotic hepatocellular dysfunction, jeopardizing homeostasis of the detoxification capacity and organic anion metabolism of the post-cold ischemic grafts.
BACKGROUND: Small-for-size grafts often cause persistent conjugated hyperbilirubinemia in the recipient after adult-to-adult living donor liver transplantation, but the cause has not yet been clarified. In physiologic status, bilirubin is excreted from hepatocytes to the bile canaliculus by means of multidrug resistance protein (MRP) 2 and, in particular circumstances, by means of MRP3 to the sinusoidal space. The aim of this study was to research whether there is any change in bilirubin excretion pattern during liver regeneration with reference to expression of MRP2 and MRP3. METHODS: Sprague-Dawley rats underwent sham operation (n=37), 70% hepatectomy (n=38), or 90% hepatectomy (n=37). The degree of liver regeneration, total and direct bilirubin, protein synthesis, and interleukin (IL)-6 were serially assessed. Expression of MRP2 and MRP3 were semiquantified by Western blotting. RESULTS: The proliferating cell nuclear antigen labeling index indicated rapid liver regeneration after 70% and 90% hepatectomy. Serum levels of total and direct bilirubin increased significantly (P<0.05), and conjugated hyperbilirubinemia was proved only in the 90% hepatectomy group. Coagulation factor VII dipped but increased as early as 12 to 24 hr postoperatively in both hepatectomy groups. Plasma IL-6 levels were significantly increased in the 90% hepatectomy group (P<0.05). Expression of MRP2 was decreased and MRP3 was expressed at 36 and 72 hr postoperatively in the 90% hepatectomy group, whereas no change was observed in MRP expression in the 70% hepatectomy group. CONCLUSIONS: During liver regeneration after critical hepatectomy such as 90% hepatectomy, decrease of MRP2 and expression of MRP3 may play an important role in postoperative hyperbilirubinemia.
4-hydroxynonenal (4HNE) is a major product of peroxidative membrane lipid destruction and exerts a variety of deleterious actions through formation of covalent adducts with cellular nucleophiles. Consequently, a number of cellular enzyme systems exist that are capable of detoxifying this reactive aldehyde by oxidation, reduction, or conjugation with glutathione. In this investigation we characterize the multidrug resistance-associated protein 2 (MRP2) as the primary transmembrane transport protein in hepatocytes responsible for extracellular export of 4HNE-glutathione conjugate (HNE-SG) from the intracellular site of its formation. Suspensions of freshly isolated hepatocytes (10(6) cells/ml) prepared from either wild-type (WT) Wistar rats or TR(-) rats possessing a mutated Mrp2 gene were incubated with 4HNE (50 nmol/10(6) cells). The formation of 4HNE metabolites, 4-hydroxynonenoic acid (HNA) and HNE-SG, was quantified in the intracellular and extracellular fractions. These studies demonstrated that freshly isolated hepatocytes from both WT and TR(-) rats formed and exported the oxidized metabolite (HNA) to similar extents. Likewise, both populations of hepatocytes displayed nearly identical rates of glutathione conjugation with 4HNE. However, the rate of HNE-SG export from TR(-) hepatocytes was approximately fourfold less than that of WT hepatocytes. In TR(-) hepatocytes, HNE-SG accumulated and remained predominantly intracellular throughout the time course, suggesting an absence of compensatory export by other hepatocellular transporters. In conclusion, these data demonstrate that although WT and TR(-) hepatocytes are similar in their conjugative and oxidative metabolism of 4HNE, export of 4HNE-SG is mediated by the MRP2 transporter, a transport system distinct from that involved in HNA efflux.
PURPOSE: Different adenosine triphosphate (ATP)-driven multidrug transporters have been described to be expressed in the luminal membrane of blood-brain barrier (BBB) endothelial cells. At this site, multidrug transporters have been suggested to restrict penetration of drugs into the brain. Increasing evidence suggests that overexpression of different multidrug transporters occurs in the region of the epileptic focus of pharmacoresistant epilepsy patients. Based on the assumption that antiepileptic drugs (AEDs) are substrates of these transporters, this overexpression may limit access of AEDs to epileptic neurons and may contribute to drug-refractoriness. In a recent study, overexpression of multidrug resistance protein 2 (ABCC2; MRP2) was reported in BBB endothelial cells of epileptic focal tissue from pharmacoresistant patients. With brain microdialysis, we recently demonstrated that the AED phenytoin is subject to transport by ABCC2 at the BBB, whereas phenobarbital does not seem to be a substrate of ABCC2. METHODS: We investigated whether ABCC2 is functionally involved in transport of the AEDs carbamazepine (CBZ), lamotrigine (LTG), and felbamate (FBM) across the BBB. The distribution of these AEDs into the brain of ABCC2-deficient TR- rats was determined. RESULTS: AED concentrations in plasma and brain extracellular space of these mutant rats did not differ significantly from those of rats of the corresponding background strain. In the amygdala-kindling model of epilepsy, the anticonvulsant efficacy of LTG and FBM was comparable in both groups of rats. In contrast, CBZ exhibited a higher anticonvulsant activity in kindled ABCC2-deficient rats as compared with nonmutant rats. CONCLUSIONS: In this present study, the microdialysis results gave no evidence that ABCC2 function modulates entry of CBZ, LTG, and FBM into the CNS of naive rats. However, ABCC2 deficiency was associated with an increased anticonvulsant response of CBZ in the kindling model. Future investigations are planned to identify the underlying mechanism for this difference, clarifying whether a pharmacokinetic difference is detectable only when brain access of CBZ is compared in kindled ABCC2-deficient rats and kindled nonmutant rats, which may have an increased expression of ABCC2 in response to seizures. The data substantiate that ABCC2-deficient TR- rats are a useful tool for defining the role of ABCC2 for transport of AEDs, and give evidence that the use of kindled TR- rats may provide important supplementary information.
Although carbon monoxide (CO) has been reported to protect against hepatobiliary dysfunction, mechanisms for its actions remain unknown. This study aimed to examine actions of physiologically relevant concentrations of CO on biliary excretion. The effects of transportal administration of CO on bile output and constituents were examined in perfused rat livers. In livers of fed rats, CO regulated bile output biphasically in a dose-dependent manner; transportal administration of CO at 4 micro mol/L stimulated bile output by 10%. Under these circumstances, CO increased paracellular junctional permeability and consequently decreased biliary excretion of bile salts. Choleresis elicited by 4 micro mol/L CO coincided with significant increases in biliary excretion of bilirubin-IXalpha and glutathione. The CO-induced choleresis occurred independently of cyclic GMP, coincided with elevated excretion of K(+) and HCO(3)(-), and was abolished by tetraethylammonium, suggesting stimulatory effects of the gas on potassium channels. CO-mediated choleresis and increased excretion of organic anions appeared to be mediated by mrp2, because Eisai hyperbilirubinemia rats, which genetically lack the transporter, did not exhibit choleresis upon the CO administration. These results suggest that CO stimulates mrp2-dependent excretion of bilirubin-IXalpha through mechanisms involving potassium channels, serving as a cooperator standing behind the heme oxygenase reaction to facilitate hepatic heme detoxification.
Hepatobiliary transporters are down-regulated in toxic and cholestatic liver injury. Cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) are attributed to mediate this regulation, but their particular contribution in vivo is still unknown. Thus, we studied the molecular mechanisms by which Ntcp, Oatp1, Oatp2, and Mrp2 are regulated by proinflammatory cytokines during liver injury. Rats were injected intraperitoneally with either carbon tetrachloride or endotoxin. Inactivation of TNF-alpha and IL-1 beta was achieved by repetitive intraperitoneal injection of etanercept and anakinra, respectively. Messenger RNA (mRNA) levels of transporters and binding activities as well as nuclear protein levels of Ntcp, Oatp2, and Mrp2 transactivators were determined 20 to 24 hours later. In contrast to IL-1 beta, TNF-alpha inactivation alone fully prevented down-regulation of Ntcp, Oatp1, and Oatp2 mRNA as well as reduced binding activity of hepatocyte nuclear factor 1 (HNF-1) in CCl(4)-induced toxic injury. In endotoxemia, down-regulation of Mrp2, and partially in case of Ntcp, could be prevented by IL-1 beta but not TNF-alpha blockade. However, inactivation of either cytokine led to preservation of HNF1 and partially of retinoid X receptor/retinoic acid receptor (RXR/RAR) binding activity. No effect of anticytokines was seen on pregnane X receptor (PXR) and constitutive androstane receptor (CAR) binding activity as well as nuclear protein mass. In conclusion, TNF-alpha represents the master cytokine responsible for HNF1-dependent down-regulation of Ntcp, Oatp1, and Oatp2 in CCl(4)-induced toxic liver injury. IL-1 beta predominates in a complex signaling network of Ntcp and Mrp2 regulation in cholestatic liver injury. In contrast to in vitro studies, HNF1 and RXR/RAR-independent mechanisms appear to be more important in regulation of Mrp2 and Ntcp gene expression in endotoxemia.
The blood-brain barrier (BBB) is a physical and metabolic barrier between the brain and the systemic circulation, which functions to protect the brain from circulating drugs, toxins, and xenobiotics. ATP-dependent multidrug transporters such as P-glycoprotein (Pgp; ABCB1), which are found in the apical (luminal) membranes of brain capillary endothelial cells, are thought to play an important role in BBB function by limiting drug penetration into the brain. More recently, the multidrug resistance protein MRP2 (ABCC2) has been found in the luminal surface of brain capillary endothelium of different species, including humans. In endothelial cells from patients with drug-resistant epilepsy, MRP2 was shown to be overexpressed, indicating that it may be critically involved in multidrug resistance of such patients. However, the role of MRP2 in drug disposition into the brain is defined poorly. Herein, we used different strategies to study the contribution of MRP2 to BBB function. First, the MRP inhibitor probenecid was shown to increase extracellular brain levels of the major antiepileptic drug phenytoin in rats, indicating that phenytoin is a substrate of MRP2 in the BBB. This was substantiated by using MRP2-deficient TR- rats, in which extracellular brain levels of phenytoin were significantly higher compared with the normal background strain. In the kindling model of epilepsy, coadministration of probenecid significantly increased the anticonvulsant activity of phenytoin. In kindled MRP2-deficient rats, phenytoin exerted a markedly higher anticonvulsant activity than in normal rats. These data indicate that MRP2 substantially contributes to BBB function.
Bile formation depends on the active secretion of bile salts and other biliary constituents by specific transporters. Recently two major transporters that contribute to bile formation, the bile salt export pump (Bsep) and multidrug resistance protein-2 (Mrp2), have been cloned. The goal of the present study was to define the expression of Bsep and Mrp2 during rat liver development. mRNA expression as assessed by Northern blot and RT-PCR was higher for Mrp2 (40% of adult) at 21 d fetal age relative to Bsep (<20% of adult). The levels of Mrp2 mRNA increased to approximately 50% of adult at 1 d of life and then rapidly increased to adult levels by 1-3 wk. Nuclear run-on assays for Bsep and Mrp2 showed minimal transcription during fetal life with an increase in transcription in the postnatal period. A different pattern of expression was observed for both Mrp2 and Bsep proteins. During fetal life, there was low expression of Mrp2 and Bsep proteins (<20% of adult) with a gradual increase neonatally reaching adult levels at 4 wk. Thus, we noted a temporal delay between the maximal expression of the mRNA (1-3 wk) and protein (4 wk) for Bsep and Mrp2. These results show that 1) expression (of mRNA and protein) of canalicular transporters is developmentally regulated by both transcriptional and posttranscriptional mechanisms and 2) Mrp2 and Bsep gene expression (mRNA) are differentially regulated.
Liver damage activates processes aimed at repairing damage; simultaneously, liver functions required for survival must be maintained. The expression of genes responsible for both in rat models of lethal (lipopolysaccharide, 90% hepatectomy, and d-galactosamine) and nonlethal (turpentine, 70% hepatectomy, and acetaminophen) liver damage and stress was measured at 3, 6, 12, and 24 h after the intervention and quantitated as the area between the control curves and the test curves (AUC). The expression of genes for cell division and remodeling was upregulated most in the lethal models. The expression of most liver-specific function genes was reduced. Positive AUC was found for ARG, ASL, CPT1, Mdr1b, Mdr2, and PEPCK. It is concluded that a high expression of genes for repair of liver damage is associated with reduced expression of genes for several liver-specific functions, possibly reflecting a limited capacity for transcriptional activity. Maintained or increased expression of selected function genes indicates that the corresponding functions have high priority. The liver sustains metabolic homeostasis ensuring that other organs in the body function normally. Simultaneously, the processes required for the integrity of its own structure and function are maintained as a result of regulated expression of the genes that produce the proteins needed to perform both set of functions.
Adenosine triphosphate binding cassette family transport proteins are important organic ion transporters in hepatocytes but these molecules may also exhibit other functions. In the present study we have measured the effects of substrates of the canalicular organic ion transporter multidrug resistance associated protein 2 (Mrp2) on chloride channel activation and cell volume regulation. We found that substrates such as leukotriene D(4), 17-beta-estradiol glucuronide, and the leukotriene inhibitor MK-571 accelerated the activation of chloride channels by cell swelling and activated chloride channels in cytokine-pretreated hepatocytes. Two conjugated estrogens that are not Mrp2 substrates did not produce this effect. Hepatocytes derived from a strain of transport-deficient rats (TR(-)), which lack Mrp2 expression, showed none of these substrate effects. Coincident with their ability to activate channels, the Mrp2 substrates increased the rate of volume regulatory decrease by approximately 50% (P <.01), confirming that enhanced channel activation under this condition stimulated volume regulation. In TR-hepatocytes the Mrp2 substrate had no effect on volume regulation. In conclusion, Mrp2 plays a role in regulation of chloride channel function by reducing the lag time necessary for channel activation and consequently accelerating the process of cell volume regulation. Substrates of Mrp2 affect the ability of the protein to interact with chloride channels. These findings represent an alternative function of Mrp2 in hepatocytes.
Regulation of bilirubin glucuronide transporters during hyperbilirubinemia in hepatic and extrahepatic tissues is not completely clear. In the present study, we evaluated the regulation of the bilirubin glucuronide transporters, multidrug resistance-associated proteins (MRP)2 and 3, in rats with obstructive jaundice. Bile duct ligation (BDL) or sham operation was performed in Wistar rats. Liver and kidneys were removed 1, 3, and 5 days after BDL (n = 4, in each group). Serum and urine were collected to measure bilirubin levels just before animal killing. MRP2 And MRP3 mRNA expressions were determined by real-time RT-PCR. Protein expression of MRP2 and MRP3 was determined by Western blotting. Renal MRP2 function was evaluated by para-aminohippurate (PAH) clearance. The effect of conjugated bilirubin, unconjugated bilirubin, human bile, and sulfate-conjugated bile acid on MRP2 gene expression was also evaluated in renal and hepatocyte cell lines. Serum bilirubin and urinary bilirubin excretion increased significantly after BDL. In the liver, the mRNA expression of MRP2 decreased 59, 86, and 82%, and its protein expression decreased 25, 74, and 93% compared with sham-operated animals after 24, 72, and 120 h of BDL, respectively. In contrast, the liver expression of MRP3 mRNA increased 138, 2,137, and 3,295%, and its protein expression increased 560, 634, and 612% compared with sham-operated animals after 24, 72, and 120 h of BDL, respectively. On the other hand, in the kidneys, the mRNA expression of MRP2 increased 162, 73, and 21%, and its protein expression increased 387, 558, and 472% compared with sham-operated animals after 24, 72, and 120 h of BDL, respectively. PAH clearance was significantly increased after BDL. The mRNA expression of MRP2 increased in renal proximal tubular epithelial cells after treatment with conjugated bilirubin, sulfate-conjugated bile acid or human bile. Upregulation of MRP2 in the kidneys and MRP3 in the liver may be a compensatory mechanism to improve bilirubin clearance during obstructive jaundice.
Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.
ATP-dependent transport of glutathione and glucuronate conjugates from hepatocytes into bile is mediated by a distinct member of the ATP-binding cassette superfamily. We have cloned and sequenced the canalicular isoform of the multidrug resistance protein from rat liver, and termed it cMrp. This membrane glycoprotein is composed of 1541 amino acids with an identity of 47.8% with the human multidrug resistance protein (MRP) and of 41.9% with the yeast cadmium factor (YCF1). The carboxyl-terminal 130 amino acids of the human hepatocyte canalicular isoform of MRP (cMRP) were 80.2% identical with rat cMrp. cMrp was not expressed in the liver of two mutant rat strains, the Eisai hyperbilirubinemic rat and the GY/TR- mutant, which are deficient in the ATP-dependent transport of conjugates across the canalicular membrane. Immunoblotting using an antibody raised against the carboxyl terminus of cMrp detected the glycoprotein of about 190 kDa only in the canalicular membrane from normal liver. Double immunofluorescence and confocal laser scanning microscopy localized cMrp exclusively to the canalicular membrane domain of hepatocytes and demonstrated its loss in the hyperbilirubinemic mutant rat. The results identify cMrp as a canalicular transport protein with a novel sequence and with a function similar to the one of the MRP.
The human Dubin-Johnson syndrome and its animal model, the TR(-) rat, are characterized by a chronic conjugated hyperbilirubinemia. TR(-) rats are defective in the canalicular multispecific organic anion transporter (cMOAT), which mediates hepatobiliary excretion of numerous organic anions. The complementary DNA for rat cmoat, a homolog of the human multidrug resistance gene (hMRP1), was isolated and shown to be expressed in the canalicular membrane of hepatocytes. In the TR(-) rat, a single-nucleotide deletion in this gene resulted in a reduced messenger RNA level and absence of the protein. It is likely that this mutation accounts for the TR(-) phenotype.