Functional analysis of dog multidrug resistance-associated protein 2 (Mrp2) in comparison with rat Mrp2.

We investigated whether the species difference in the biliary excretion activity of some Mrp2 substrates was attributable to the intrinsic transport potential or the expression level of Mrp2, especially in rat and dog. Dog Mrp2 cDNA was isolated from beagle dog liver, and a vesicle transport study was performed using recombinant rat and dog Mrp2 expressed in insect Sf9 cells. The ATP-dependent transport of 17beta-estradiol 17-(beta-D-glucuronide) ([3H]E(2)17betaG) and leukotriene C4 ([3H]LTC4), normalized by the absolute protein expression level, was similar in both Mrp2s. The Mrp2 protein expression in dog liver was only 10% of that in rat liver and was comparable with the reported difference in the biliary excretion clearance of temocaprilat as Mrp2 substrate. In contrast to LTC4, unique transport kinetics for E(2)17betaG were evident in dog Mrp2. In addition to the high-affinity site with a K(m) value of 3.25 +/- 0.10 microM, which is similar to that in rat Mrp2 (4.81 +/- 1.21 microM), dog Mrp2 has an additional low-affinity site (>75 microM), which makes a major contribution to the transport of E(2)17betaG (65% of the total transport capacity at tracer concentration). In summary, the difference in the biliary excretion activity of Mrp2 substrates between rat and dog depends on the Mrp2 protein expression level rather than the intrinsic transport activity of the transporter molecules. The unique transport properties of glucuronide conjugates by dog Mrp2 may lead to the species difference involving the drug-drug interaction or drug-induced hyperbilirubinemia on the bile canalicular membrane.     

Characterization of transport protein expression in multidrug resistance-associated protein (Mrp) 2-deficient rats.

Multidrug resistance-associated protein (Mrp) 2-deficient transport-deficient (TR(-)) rats, together with their transport-competent Wistar counterparts (wild type), have been used to examine the contribution of Mrp2 to drug disposition. However, little is known about potential variation in expression of other transport proteins between TR(-) and wild-type rats or whether these differences are tissue-specific. Sections of liver, kidney, brain, duodenum, jejunum, ileum, and colon were obtained from male TR(-) and wild-type Wistar rats. Samples were homogenized in protease inhibitor cocktail and ultracentrifuged at 100,000g for 30 min to obtain membrane fractions. Mrp2, Mrp3, Mrp4, P-glycoprotein, sodium-dependent taurocholate cotransporting polypeptide, organic anion transporting polypeptides 1a1 and 1a4, bile salt export pump, breast cancer resistance protein, ileal bile acid transporter, UDP-glucuronosyl transferase (UGT1a), glyceraldehyde-3-phosphate dehydrogenase, and beta-actin protein expression were determined by Western blot. Mrp3 was significantly up-regulated in the liver ( approximately 6-fold) and kidney ( approximately 3.5-fold) of TR(-) rats compared with wild-type controls. Likewise, the expression of UGT1a enzymes was increased in the liver and kidney of TR(-) rats by approximately 3.5- and approximately 5.5-fold, respectively. Interestingly, Mrp3 expression was down-regulated in the small intestine of TR(-) rats, but expression was similar to wild type in the colon. Mrp4 was expressed to varying extents along the intestine. Expression of some transport proteins and UGT1a enzymes differ significantly between TR(-) and wild-type rats. Therefore, altered drug disposition in TR(-) rats must be interpreted cautiously because up- or down-regulation of other transport proteins may play compensatory roles in the presence of Mrp2 deficiency.     

Role of glycosylation in trafficking of Mrp2 in sandwich-cultured rat hepatocytes

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

Functional analysis of mouse and monkey multidrug resistance-associated protein 2 (Mrp2).

We investigated the intrinsic transport activity of mouse and monkey Mrp2 and compared it with that of rat and dog Mrp2 reported previously. Mrp2 cDNAs were isolated from BALB/c and Macaca fascicularis liver, respectively, and vesicle transport studies were performed using recombinant Mrp2s expressed in insect Sf9 cells. ATP-dependent transport of [3H]leukotriene C4 (LTC4), [3H]17beta-estradiol 17-(beta-D-glucuronide) (E217betaG), [3H]bromosulfophthalein (BSP), and [3H]cholecystokinin octapeptide (CCK-8) were readily detected for all Mrp2s. A species difference in the intrinsic transport activity was apparent for LTC4 (monkey > mouse, dog > rat) and BSP (rat, dog, monkey > mouse). In addition to the difference in the transport activity, complex kinetic profiles were also evident in CCK-8, where a cooperative transport site was observed. Moreover, the transport of [3H]E217betaG by mouse and monkey Mrp2 was quite different from that of rat and dog Mrp2 in that 1) there was practically only nonsaturable uptake for [3H]E217betaG and 2) 4-methylumbelliferon glucuronide (Mrp2 modulator) showed a concentration-dependent stimulatory effect on the transport of [3H]E217betaG in mouse and monkey Mrp2, whereas rat and dog transport activity was inhibited by the modulator. In conclusion, although the substrate specificity is similar, the intrinsic transport activity differs from one species to another. This is due not only to the difference in the Km and Vmax values, but also the qualitatively different mode of substrate and modulator recognition exhibited by different species.     

Hormonal regulation of renal multidrug resistance-associated proteins 3 and 4 (Mrp3 and Mrp4) in mice

Multidrug resistance-associated proteins 3 and 4 (Mrp3 and Mrp4) are expressed at much higher levels in female than male kidney. Sex steroids and sex-specific growth hormone (GH) secretion patterns often mediate gender-predominant gene expression. Thus, three models were used to investigate potential endocrine regulation of Mrp3 and Mrp4: (1) gonadectomized (GNX) mice with 17beta-estradiol (E2) or 5alpha-dihydroxytestosterone (DHT) replacement; (2) hypophysectomized (HPX) mice receiving E2, DHT, or simulated male-pattern (MP) or female-pattern (FP) GH secretion; (3) lit/lit mice, which have a spontaneous mutation in the growth-hormone releasing-hormone (GHRH) receptor, with simulated MP- or FP-GH secretion. GNX and HPX decreased Mrp3 mRNA levels compared with intact females. In both respective models E2 administration increased Mrp3 expression in GNX and HPX mice. DHT markedly repressed Mrp3 from GNX+placebo levels, however, this was not observed in the HPX model. In lit/lit mice, Mrp3 expression was lower than in wild-type controls, and MP-GH and FP-GH simulation slightly increased Mrp3 expression. Whereas GNX increased Mrp4 in males to female levels, HPX actually increased Mrp4 expression in both genders +375% and +66%, respectively. In both models DHT markedly repressed Mrp4. Furthermore, Mrp4 was higher in lit/lit than wild-type male mice, and simulation of MP-GH secretion suppressed female-predominant Mrp4 expression. In conclusion, these data indicate that E2 contributes to higher Mrp3 mRNA expression in females, yet a role for androgens in Mrp3 repression cannot be discounted. In contrast, Mrp4 mRNA is higher in females due to repression by both DHT and MP-GH secretion in males.     

基于多药耐药相关蛋白2(Abcc2/Mrp2)在大鼠肾脏表达性别差异的氧氟沙星药动学变化(英文)

本研究旨在探讨基于多药耐药相关蛋白2(Abcc2/Mrp2)在大鼠肾脏表达性别差异的氧氟沙星的药代动力学变化。采用高效液相色谱法(HPLC)分别测定经尾静脉注射给予氧氟沙星(30 mg.kg1)后大鼠血浆和尿液中氧氟沙星的浓度;通过免疫组化法和流式细胞术分别对雄性和雌性大鼠肾脏Mrp2的表达进行定性和定量分析。结果表明在雄性大鼠体内,氧氟沙星药时曲线下面积(AUC)明显小于雌性大鼠,而尿排总量明显大于雌性大鼠;Mrp2在雄性大鼠肾脏中的表达显著高于雌性大鼠。因此,氧氟沙星药动学性别差异可能是由Mrp2在雄性和雌性大鼠肾脏中的表达差异所引起的。     

Effect of culture conditions on the expression and function of Bsep, Mrp2, and Mdr1a/b in sandwich-cultured rat hepatocytes

Rat hepatocytes cultured in a sandwich configuration form functional canalicular networks. The influence of extracellular matrix configuration, medium composition, and confluency on the expression and function of Bsep, Mrp2, and Mdr1a/b in sandwich-cultured (SC) rat hepatocytes was examined. Primary rat hepatocytes were: (1) maintained in various extracellular matrix sandwich configurations, (2) cultured in Dulbecco's modified Eagle's medium (DMEM), Modified Chee's medium (MCM) or Williams' E medium (WME), and/or (3) plated at decreasing cell density. Bsep, Mrp2, and Mrdr1a/b expression in day 4 SC rat hepatocytes was assessed by Western blot; function was measured by accumulation of taurocholate, 5(and 6)-carboxy-2',7'-dichlorofluorescein, and rhodamine 123, respectively, in canalicular networks. In general, the extracellular matrix conditions examined resulted in similar protein expression and function. Function of Bsep, Mrp2, and Mdr1a/b was higher in SC rat hepatocytes maintained in DMEM or WME. Mrp2 and Mdr1a/b expression, representative of total cellular content, did not always correlate directly with function, which should be reflective of canalicular membrane expression. Mrp2 expression decreased significantly as cell density decreased in SC hepatocytes. Low plating density in Biocoat plates resulted in poor canalicular network formation and reduced function of Mrp2 and Mdr1a/b. Expression and/or function of Mrp2 and Mdr1a/b in rat hepatocytes cultured in a sandwich configuration may be influenced by plating density and media type.     

Effect of ursodeoxycholic acid treatment on the expression and function of multidrug resistance-associated protein 2 in rat intestine

Effect of ursodeoxycholic acid (UDCA) treatment on the expression and function of intestinal multidrug resistance-associated protein (Mrp) 2 was examined in rats. When rats were orally administered 0.5% UDCA solution for 6 days, mRNA and protein levels of Mrp2 in the intestine were increased about twofold compared with those in untreated rats. In in vitro everted sac study, Mrp2-mediated efflux of 2,4-dinitrophenyl-S-glutathione (DNP-SG) to the mucosal surface was shown to be increased by UDCA treatment. In vivo intestinal exsorption clearance of DNP-SG was also increased by UDCA treatment. In addition, in situ intestinal absorption of methotrexate, a substrate of Mrp2, was decreased by the treatment. These results indicate that the expression and function of intestinal Mrp2 is up-regulated by oral administration of UDCA. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2822–2831, 2009     

Tissue distribution and hepatic and renal ontogeny of the multidrug resistance-associated protein (Mrp) family in mice.

Analysis of the mouse genome has revealed eight multidrug resistance-associated (Mrp) transporters, with mouse homologs for all human MRPs except MRP8. Whereas MRP expression in tissues of humans and rats has been examined, no characterization exists for mice. Furthermore, the ontogeny of mouse Mrps is unknown, and such knowledge may be helpful in understanding age-related pharmacokinetics. Therefore, the purpose of this study was to quantitatively determine 1) expression of the Mrp family in 12 different tissues, 2) gender variations in Mrp expression in liver and kidney, and 3) whether Mrp expression is altered during development. Highest expression of the Mrp family members is as follows: Mrp1 in testes, ovary, and placenta; Mrp2 in intestine, followed by liver and kidney; Mrp3 in large intestine; Mrp4 in kidney; Mrp5 in brain, followed by lung and stomach; Mrp6 in liver; Mrp7 in testes, intestine, and kidney; and Mrp9 solely in testes. Gender differences in Mrp expression were observed: Mrp1, 3, and 4 in kidney, as well as Mrp1 and 4 in liver were female-predominant. Ontogeny of the four Mrps expressed in liver was as follows: Mrp2 and Mrp4 were expressed at adult levels at birth; Mrp3 reached adult levels at day 30, and Mrp6 was not expressed until day 10. In kidney, Mrp1 and Mrp5 were expressed at adult levels at birth, whereas Mrp2, 3, 4, and 6 generally increased over time. In conclusion, marked differences in expression of the individual Mrp family members exist in various tissues, with age, and with gender.     

Effect of acetaminophen on expression and activity of rat liver multidrug resistance-associated protein 2 and P-glycoprotein

We evaluated the effect of acetaminophen (APAP), given as a single, 1g/kg body weight dose, on expression and activity of rat liver multidrug resistance-associated protein 2 (Mrp2) and P-glycoprotein (P-gp), two major canalicular drug transporters. The studies were performed 24h after administration of the drug. APAP induced an increase in plasma membrane content of Mrp2 detected by western blotting, consistent with increased detection of the protein at the canalicular level by immunoflourescence microscopy. In vivo biliary excretion of dinitrophenyl-S-glutathione, a well known Mrp2 substrate, was slightly but significantly increased by APAP, agreeing well with upregulation of the transporter. Basal biliary excretion of oxidized glutathione, an endogenous Mrp2 substrate, was also increased by APAP, likely indicating increased hepatic synthesis as a result of APAP-induced oxidative stress followed by accelerated canalicular secretion mediated by Mrp2. APAP also increased the expression of P-gp detected by western blotting and immunofluorescence microscopy as well as the in vivo biliary secretory rate of digoxin, a model P-gp substrate. Because specific APAP-conjugated metabolites are Mrp2 substrates, we postulate that induction of Mrp2 by APAP may represent an adaptive mechanism to accelerate liver disposition of the drug. In addition, increased Mrp2-mediated elimination of oxidized glutathione may be essential in maintaining the redox equilibrium in the hepatocyte under conditions of APAP-induced oxidative stress.     

Modulation of gene expression by antisense and antigene oligodeoxynucleotides and small interfering RNA

Antisense oligodeoxynucleotides, triplex-forming oligodeoxynucleotides and double-stranded small interfering RNAs have great potential for the treatment of many severe and debilitating diseases. Concerted efforts from both industry and academia have made significant progress in turning these nucleic acid drugs into therapeutics, and there is already one FDA-approved antisense drug in the clinic. Despite the success of one product and several other ongoing clinical trials, challenges still exist in their stability, cellular uptake, disposition, site-specific delivery and therapeutic efficacy. The principles, strategies and delivery consideration of these nucleic acids are reviewed. Furthermore, the ways to overcome the biological barriers are also discussed so that therapeutic concentrations at their target sites can be maintained for a desired period.     

Differential regulation of multidrug resistance-associated protein 2 (MRP2) and cytochromes P450 2B1/2 and 3A1/2 in phenobarbital-treated hepatocytes

Multidrug resistance-associated protein 2 (MRP2) is a drug efflux pump found at the biliary pole of hepatocytes. In the present study, we have investigated its expression in response to phenobarbital, a liver tumor promoter known to up-regulate hepatic cytochromes P450 (CYPs), such as CYP2B1/2 and CYP3A1/2. MRP2 mRNA and protein levels were found to be markedly increased in both primary rat and human hepatocytes exposed to phenobarbital. However, features of this up-regulation, especially the dose-response, were different from those of the induction of CYP2B1/2 and CYP3A1/2. In addition, hepatic MRP2 expression remained unaltered in rats treated by phenobarbital that, by contrast, increased CYP2B1/2 and CYP3A1/2 gene expression in the liver. Therefore, MRP2 and CYPs appeared differently regulated in response to phenobarbital in both in vivo and in vitro situations, suggesting that cellular and molecular mechanisms underlying up-regulation of MRP2 are, at least in part, unrelated to those operating for CYPs. Phenobarbital-related MRP2 induction in primary rat hepatocytes was associated with some phenotypic effects of the barbiturate, such as prolonged cell survival and inhibition of cell proliferation. Phenobarbital also inhibited growth of human hepatoma HepG(2) cells and increased their level of MRP2 gene expression. Such results may favor a putative relationship between phenobarbital-mediated MRP2 regulation in cultured liver parenchymal cells and alteration of cell cycle and survival.     

Ischemia-reperfusion of rat livers decreases liver and increases kidney multidrug resistance associated protein 2 (Mrp2).

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.     

Effect of dexamethasone treatment on the expression and function of transport proteins in sandwich-cultured rat hepatocytes.

Dexamethasone (DEX) is a well established inducer of CYP3A. These studies examined the influence of DEX treatment on transport protein expression and function in sandwich-cultured (SC) rat hepatocytes. Freshly isolated hepatocytes were cultured between two layers of gelled collagen and maintained in Dulbecco's modified Eagle's medium supplemented with DEX (0.1 microM, 0-48 h and 0.1-100 microM, 48-96 h). The expression of sinusoidal [(organic anion transporting polypeptide 1a1 (Oatp1a1), Oatp1a4, multidrug resistance-associated protein 3 (Mrp3), and Na(+)-dependent taurocholate cotransporting polypeptide (Ntcp)] and canalicular [bile salt export pump (Bsep), multidrug resistance protein 1a/b (Mdr1a/b), and Mrp2] transport proteins was determined by Western blot analysis. The accumulation and biliary excretion index (BEI; percentage of accumulated substrate in canalicular networks) of the probe substrates taurocholate (TC; 1 microM, 10 min), rhodamine 123 (Rh123; 10 microM, 30 min), and carboxy-2',7'-dichlorofluorescein (CDF; 10 microM, 10 min) were employed as measures of canalicular transport protein function in SC rat hepatocytes. DEX treatment increased CYP3A1/2, Oatp1a4, and Mrp2 expression, decreased the expression of Ntcp, and did not seem to alter the expression of Oatp1a1, Mrp3, Mdr1a/b, or Bsep. The BEI of CDF, an Mrp2 substrate, increased from 18 to 37% after DEX treatment (100 microM). The accumulation of TC, an Ntcp substrate, was reduced (<50% of control), whereas the BEI of TC, also a Bsep substrate, was unchanged. Treatment of SC rat hepatocytes with DEX resulted in alterations in the expression of CYP3A1/2 and some hepatic transport proteins. Modest alterations in hepatic transport protein function were consistent with changes in protein expression.     

Key determinants of the circulatory exposure of organic anions: differences in hepatic uptake between multidrug resistance-associated protein 2 (Mrp2)-deficient rats and wild-type rats

Abstract

1.?Raloxifene-6-glucuronide (R6G) is a substrate of rat multidrug resistance-associated protein 2 (Mrp2), a transporter responsible for biliary excretion of organic anions.

2.?Pharmacokinetic modeling of R6G in Eisai hyperbilirubinemic rats (EHBRs), hereditary Mrp2-deficient rats, and wild-type Sprague–Dawley rats (SDRs) indicated that reduction in not only biliary excretion but also hepatic uptake of R6G influenced low clearance in EHBRs.

3.?An integration plot study demonstrated that the hepatic uptake of R6G was 66% lower in EHBRs than that in SDRs. A reduction was observed for the other Mrp2 substrate Valsartan (95% lower) but not for estradiol-17β-glucuronide (E₂17βG). This variation may be associated with the difference in substrate specificity of transporters and/or inhibition of hepatic uptake of organic anions by endogenous substances such as bilirubin glucuronides.

4.?In conclusion, incidental alteration of the hepatic uptake of organic anions should be considered as an explanation of their enhanced systemic exposure in EHBRs.