Involvement of multidrug resistance-associated proteins in regulating cellular levels of (-)-epigallocatechin-3-gallate and its methyl metabolites

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol of green tea, has many interesting biological activities. The uptake of EGCG and involvement of specific efflux pumps were studied in MDCKII cells transfected with hPgp, hMRP1, and hMRP2 genes. Total cell associated [3H]EGCG increased 7-fold in the presence of the MRP inhibitors, indomethacin and probenecid, in MDCKII/MRP1 cells, compared to a 2-fold increase in wild-type cells. Intracellular levels of EGCG, 4(")-O-methyl EGCG, and 4('),4(")-di-O-methyl EGCG were increased by 13-, 11-, and 3-fold, respectively, by indomethacin in MDCKII/MRP1 cells. Accumulation of EGCG and its methyl metabolites was also increased approximately 10-fold in the presence of MK-571 in MDCKII/MRP2 cells. Co-treatment with isoflavones, curcumin and tetrahydrocurcumin, increased [3H]EGCG accumulation significantly in MDCKII/MRP1 and HT-29 cells. The results indicate that EGCG and its methyl metabolites are substrates for MRP1 and MRP2, but not for Pgp. MRP type efflux pumps may limit the bioavailability of EGCG.     

In vivo relevance of Mrp2-mediated biliary excretion of the Amanita mushroom toxin demethylphalloin

To determine which efflux carriers are involved in hepatic phalloidin elimination, hepatobiliary [(3)H]-demethylphalloin (DMP) excretion was studied in normal Wistar rats and in Mrp2 deficient TR(-) Wistar rats as well as in normal wild-type FVB mice, Mdr1a,b(-/-) knockout mice, and Bcrp1(-/-) knockout mice by in situ bile duct/gallbladder cannulation. A subtoxic dose of 0.03 mg DMP/kg b.w. was used, which did not induce cholestasis in any tested animal. Excretion of DMP into bile was not altered in Mdr1a,b(-/-) mice or in Bcrp1(-/-) mice compared with wild-type FVB mice. Whereas 17.6% of the applied dose was excreted into bile of normal Wistar rats, hepatobiliary excretion decreased to 7.9% in TR(-) rats within 2 h after intravenous application. This decrease was not due to reduced cellular DMP uptake, as shown by normal expression of Oatp1b2 in livers of TR(-) rats and functional DMP uptake into isolated TR(-) rat hepatocytes. Tissue concentrations of phalloidin were also not altered in any of the transgenic mice. Interestingly, the decrease of biliary DMP excretion in the TR(-) rats was not followed by any increase of phalloidin accumulation in the liver but yielded a compensatory excretion of the toxin into urine, indicating that hepatocytes of TR(-) rats expelled phalloidin back into blood circulation.     

In vivo relevance of Mrp2-mediated biliary excretion of the Amanita mushroom toxin demethylphalloin

To determine which efflux carriers are involved in hepatic phalloidin elimination, hepatobiliary [³H]-demethylphalloin (DMP) excretion was studied in normal Wistar rats and in Mrp2 deficient TR(−) Wistar rats as well as in normal wild-type FVB mice, Mdr1a,b(−/−) knockout mice, and Bcrp1(−/−) knockout mice by in situ bile duct/gallbladder cannulation. A subtoxic dose of 0.03 mg DMP/kg b.w. was used, which did not induce cholestasis in any tested animal. Excretion of DMP into bile was not altered in Mdr1a,b(−/−) mice or in Bcrp1(−/−) mice compared with wild-type FVB mice. Whereas 17.6% of the applied dose was excreted into bile of normal Wistar rats, hepatobiliary excretion decreased to 7.9% in TR(−) rats within 2 h after intravenous application. This decrease was not due to reduced cellular DMP uptake, as shown by normal expression of Oatp1b2 in livers of TR(−) rats and functional DMP uptake into isolated TR(−) rat hepatocytes. Tissue concentrations of phalloidin were also not altered in any of the transgenic mice. Interestingly, the decrease of biliary DMP excretion in the TR(−) rats was not followed by any increase of phalloidin accumulation in the liver but yielded a compensatory excretion of the toxin into urine, indicating that hepatocytes of TR(−) rats expelled phalloidin back into blood circulation.     

Prolonged blood glucose reduction in mrp-2 deficient rats (GY/TR(-)) by the glucose-6-phosphate translocase inhibitor S 3025

Chlorogenic acid derivatives are potent inhibitors of hepatic glucose production by inhibition of the glucose-6-phosphate translocase component of the hepatic glucose-6-phosphatase system. The pharmacological proof of concept was clearly demonstrated during i.v. infusion of potent derivatives (S 4048, S 3483) in rats. However, the blood glucose lowering effect of S 4048 after bolus i.v. injection lasted only 60-90 min. Plasma clearance of S 4048 was very high, and the parent compound was rapidly and efficiently excreted into the bile of Wistar and GY/TR(-) rats, indicating that mrp-2 was not involved in this hepatobiliary elimination process. About 72% of the total administered radioactivity appeared in the bile within 20 min after i.v. bolus injection of the radiolabeled analogue [(3)H]S 1743 in a Wistar rat. However, in GY/TR(-) rats the dicarboxylic analogue of S 4048, S 3025, was cleared from the plasma less rapidly than its parent compound and its biliary elimination was comparatively low. In contrast, S 3025 exhibited comparable pharmacokinetics and biliary elimination profile as S 4048 in Wistar rats, suggesting that biliary elimination of S 3025 is facilitated by mrp-2, functionally absent in GY/TR(-) rats. Targeting to mrp-2 resulted in a significantly prolonged reduction of blood glucose levels in GY/TR(-) rats after i.v. bolus administration of S 3025.     

Glutathione conjugation and pharmacokinetics of 2-bromo-3-phenylpropionic acid in vitro and in the rat in vivo.

Glutathione (GSH) conjugation of the chiral compound 2-bromo-3-phenylpropionic acid (BPP) was studied in vitro and in the rat in vivo. GSH conjugation of BPP, catalyzed by a mixture of glutathione-S-transferases (GST's) from rat liver cytosol in vitro, was stereoselective: at a substrate concentration of 250 microM, (R)-BPP was more rapidly conjugated than (S)-BPP (R/S-ratio = 2.6). The blood elimination kinetics of the separate BPP enantiomers and the biliary excretion kinetics of the corresponding GSH conjugates were studied in the rat in vivo after administration of (R)- or (S)-BPP at a dose level of 50 mumol/kg. Elimination of (R)-BPP from blood was faster than that of (S)-BPP: half lives were 9 +/- 2 min for (R)-BPP and 13 +/- 1 min for (S)-BPP. The biliary excretion rate of the GSH conjugate of (R)-BPP declined monoexponentially, while that of the GSH conjugate of (S)-BPP displayed a biphasic profile. Half lives of excretion were 13 +/- 1 for the GSH conjugate of (R)-BPP, and 11 +/- 2 for the GSH conjugate of (S)-BPP (second phase). The first phase in the biliary excretion of the GSH conjugate of (S)-BPP could not be attributed to capacity limitation of biliary transport carriers as higher excretion rates were attained upon administration of higher doses (100 and 200 mumol/kg) of (S)-BPP). The blood elimination profiles of (R)- and (S)-BPP differed greatly from the biliary excretion profiles of the corresponding GSH conjugates. This suggests that the kinetics of BPP conjugate excretion are determined by other processes than hepatic GSH conjugation.     

Transport of fluorescein in MDCKII-MRP1 transfected cells and mrp1-knockout mice.

The multidrug resistant-associated protein 1 (MRP1) is a membrane-bound transport protein that is involved in the efflux of organic anions and has been implicated in multidrug resistance in cancer. MRP1 has also been reported to be ubiquitously expressed in normal tissues, including the brain. The presence of functional organic anion transporters in the blood-brain and blood-CSF barriers that influence the distribution of various compounds to the brain has long been known. The purpose of this study was to examine the role of MRP1 in the brain distribution of a model organic anion, fluorescein. The substrate specificity of MRP1 for fluorescein was initially determined by examining the accumulation of fluorescein in MDCKII MRP1-transfected cells. The distribution of fluorescein in the brain was then examined in wild-type and mrp1 gene knockout mice. The results show that in MDCKII MRP1-transfected cells, the accumulation of fluorescein was significantly lower (about 40% lower) than that in wild-type MDCKII cells. MRP1 inhibitors such as probenecid, MK-571, and LY402913 enhanced fluorescein accumulation in MDCKII MRP1-transfected cells to a greater extent than in wild-type MDCKII cells. In an in vivo study, after intravenous injection of fluorescein, the fluorescein brain-to-plasma concentration ratio in mrp1 knockout mice was not significantly different than that in wild-type mice. However, when probenecid was co-administered with fluorescein in wild-type mice, the fluorescein brain-to-plasma ratio was significantly increased (1.5-fold). These findings suggest that fluorescein is a substrate for MRP1. Furthermore, the in vivo study also suggests that MRP1 has a limited role in the transport and distribution of fluorescein in the brain. Therefore, other organic anion transport proteins, including the various isoforms of the MRP family, may be responsible for the accumulation and transport of organic anions in the brain.     

Biliary excretion of flavopiridol and its glucuronides in the isolated perfused rat liver: role of multidrug resistance protein 2 (Mrp2)

Flavopiridol (FLAP) is a novel anticancer agent that is extensively glucuronidated in patients. Biliary excretion is the main elimination pathway of FLAP conjugates responsible for enterohepatic recirculation and for the main side effect diarrhea. To investigate the hepatic transport system for FLAP glucuronides, livers of Wistar and Mrp2-deficient TR- rats were perfused with FLAP (30 microM) in a single pass system. Biliary excretion and efflux into perfusate during a 60 min period greatly differ in TR- rats. While cumulative biliary excretion of M1 and M2 was significantly reduced to 4.3% and 5.4% efflux into perfusate was increased by 1.5 and 4.2-fold. This indicates that in control rats, M1 and M2 are almost exclusively eliminated into bile by Mrp2. Cumulative FLAP secretion into bile and perfusate, however, was non-significantly reduced by 36.7% and 43.2% in the mutant rat strain, suggesting that besides Mrp2, other transporters might also be involved in FLAP elimination. FLAP stimulates bile flow up to 24% in control rats, but secretion is nearly absent in TR- rats further supporting an efficient transport of FLAP glucuronides by Mrp2. FLAP (30 microM) also reversibly inhibited the Mrp2-mediated biliary elimination of bilirubin and bromsulphthalein in Wistar rats by 54% and 51%, respectively, indicating a competition with the elimination of Mrp2-specific substrates. In summary, we found that FLAP glucuronides are substrates of Mrp2 effectively inhibiting the biliary excretion of bilirubin. This may explain the increased serum bilirubin levels observed in cancer patients during FLAP therapy.     

Hepatobiliary excretion of dipyrrinone sulfonates in Mrp2-deficient (TR(-)) rats

The biliary excretion of the sodium salts of 8-(2-ethanesulfonic acid)-3-ethyl-2,7,9-trimethyl-1,10-dihydro-11H-dipyrrin-1-one (xanthosulfonic acid) and a fluorescent analogue (8-desethyl-N,N'-carbonyl-kryptopyrromethenone-8-sulfonic acid) was compared in Mrp2-deficient (TR(-)) and normal rats. Both organic anions were excreted rapidly in bile in Mrp2-deficient rats, but the biliary excretion of the fluorescent sulfonate was impaired relative to normal controls. The rat clearly has efficient Mrp2-independent mechanisms for biliary efflux of these anions that are not used by bilirubin or its mono- and diglucuronides.     

Postnatal development of the hepatobiliary transport of phenolsulfonphthalein in rats

1. The postnatal development of the biliary excretion of phenolsulfonphthalein (PSP) was studied in male Wistar rats. 2. Following i.v. injection of PSP at 200 mumol/kg body wt, a maximal biliary excretion of 175 +/- 10 nmol/min/100 g body wt and 32 +/- 5 nmol/min/100 g body wt was reached for unconjugated and conjugated PSP, respectively, in the adult group. 3. The maximal biliary excretion of conjugated PSP was significantly lower in the 20-, 30- and 40-day-old groups as compared to the adults. The excretion of unconjugated dye was also significantly lower in 20- and 30-day-old rats. 4. The postnatal development of PSP excretion was unrelated to changes in the activity of UDP-glucuronosyltransferase. The importance of other factors is also discussed.     

Biliary copper excretion capacity in intact animals: correlation between ATP7B function, hepatic mass, and biliary copper excretion

Copper toxicosis can occur in the absence of biliary copper excretion. To demonstrate whether biliary copper excretion capacity is correlated with hepatic mass and ATP7B function, we undertook studies in intact animals. Copper-histidine was injected intrasplenically after baseline bile collection, followed by measurement of copper excretion in Long-Evans Cinnamon (LEC) rats lacking atp7b function and in normal, syngeneic Long-Evans Agouti (LEA) rats. The basal biliary copper excretion was very low in LEC rats compared with LEA rats, 8+/-4 and 37+/-18 ng copper/min, respectively; p<0.05. After addition of copper, copper excretion increased significantly (by two- to five-fold) in LEA rats during the 30 minute study period, whereas LEC rats showed only a slight and transient increase in copper excretion. After one-third and two-thirds partial hepatectomy immediately before copper loading, copper excretion decreased progressively. The studies indicate that biliary copper excretion depends on hepatocyte mass and ATP7B gene function. Analysis of copper excretion with our non-radioactive method will facilitate testing of novel therapies and pathophysiological mechanisms in copper toxicity.     

Mrp2 is involved in benzylpenicillin-induced choleresis

Benzylpenicillin (PCG; 180 micromol/kg), a classic beta-lactam antibiotic, was intravenously given to Sprague-Dawley (SD) rats and multidrug resistance-associated protein 2 (Mrp2)-deficient Eisai hyperbilirubinemic rats (EHBR). A percentage of the [(3)H]PCG was excreted into the bile of the rats within 60 min (SD rats: 31.7% and EHBR: 4.3%). Remarkably, a transient increase in the bile flow ( approximately 2-fold) and a slight increase in the total biliary bilirubin excretion were observed in SD rats but not in the EHBR after PCG administration. This suggests that the biliary excretion of PCG and its choleretic effect are Mrp2-dependent. Positive correlations were observed between the biliary excretion rate of PCG and bile flow (r(2) = 0.768) and more remarkably between the biliary excretion rate of GSH and bile flow (r(2) = 0.968). No ATP-dependent uptake of [(3)H]PCG was observed in Mrp2-expressing Sf9 membrane vesicles, whereas other forms of Mrp2-substrate transport were stimulated in the presence of PCG. GSH efflux mediated by human MRP2 expressed in Madin-Darby canine kidney II cells was enhanced in the presence of PCG in a concentration-dependent manner. In conclusion, the choleretic effect of PCG is caused by the stimulation of biliary GSH efflux as well as the concentrative biliary excretion of PCG itself, both of which were Mrp2 dependent.     

Effect of ML-236B (compactin) on biliary excretion of bile salts and lipids, and on bile flow, in the rat

To assess the importance of de novo cholesterol synthesis for bile salt formation, the effects of ML-236B (an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase) on biliary excretion of bile salts and lipids were studied in rats with permanent catheters in bile duct, heart and duodenum. In rats having their bile diverted continuously for 8 days, duodenal administration of ML-236B (50 mg/kg) caused an immediate transient choleresis, which subsided after 2 h. Concomitant with the choleresis concentrations of bile salt, phospholipid and cholesterol fell, but this decrease was maintained for 6 h. Consequently, ML-236B inhibited biliary output salts and lipids from the second till the sixth hour after injection. The kinetics of biliary excretion of intravenously injected [14C]taurocholate were not affected by ML-236B administration. In rats having their biliary catheter connected to the duodenal catheter, or in rats with prolonged bile diversion but treated with mevalonolactone, ML-236B again caused a transient choleresis (having subsided after 2 h), but now did not affect biliary excretion of bile salts and lipids. It is concluded that (1) ML-236B causes a transient bile salt-independent choleresis, (2) ML-236B depresses excretion of bile salts and lipids by blocking mevalonate synthesis and not by blocking the bile salt or lipid transport, (3) biliary excretions of phospholipids and cholesterol partly depend on excretion of bile salt, and (4) in rats with a prolonged total bile diversion newly formed mevalonate is a major substrate for bile salt synthesis.     

Characterization of 5(6)-carboxy-2,'7'-dichlorofluorescein transport by MRP2 and utilization of this substrate as a fluorescent surrogate for LTC4

MRP2 (ABCC2) is an efflux transporter expressed on the apical membrane of polarized cells. This protein has a major role in the biliary elimination of toxic compounds from the liver. As MRP2 transports many endogenous compounds, including LTC4 as well as xenobiotics and toxic phase II metabolites, blockade of this transporter may cause the accumulation of these compounds in the hepatocyte, resulting in hepatotoxicity. The vesicular transport assay is a great tool to study drug-drug and drug-endogenous compound interactions of ABC transporters. In this assay, inside-out membrane vesicles are used, so the test compound can readily access the transporter. As MRP2 transports many ionic compounds that are difficult to investigate in a whole-cell system because of permeability reasons, the vesicular transport assay is a good choice for screening MRP2-mediated interactions. LTC4 is not an optimal substrate for high-throughput screening for MRP2 interactors, even though it is an important MRP2 substrate. Therefore, the transport of a drug surrogate, 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF), by MRP2 was characterized using the vesicular transport assay. The data indicate that CDCF proves to be an ideal substrate for MRP2 vesicular transport assay with its optimal detection and transport properties.     

Multidrug resistance-associated protein2 (MRP2) plays an important role in the biliary excretion of glutathione conjugates of 4-hydroxynonenal

Glutathione (GSH) conjugates of 4-hydroxy-trans-2,3-nonenal (HNE) are the final products of lipid peroxidation. In the present study, the role of multidrug resistance-associated protein 2 (MRP2) in biliary excretion of GSH conjugates of HNE (HNE-SG) was studied in vitro by using Madin-Darby canine kidney II (MDCK II) cells expressing human MRP2 and in vivo using a mutant rat strain whose MRP2 expression is defective (Eisai-hyperbilirubinemic rats [EHBR]). A high-performance liquid chromatography method was developed to assay HNE-SG conjugates. Four diastereomeric HNE-SG conjugates could be separated with this method. Three of four HNE-SG conjugates were detectable after incubation of the cell monolayers with HNE. Expression of human MRP2 resulted in a 10-fold increase in HNE-SG conjugates excretion across the apical membrane of MDCK II cells. The four HNE-SG conjugates appeared swiftly in bile from Sprague Dawley rats after intravenous administration of HNE, whereas no detectable HNE-SG conjugates were observed in the bile of EHBR. These results demonstrate the role of MRP2 in the biliary excretion of HNE-SG conjugates.     

MRP proteins as potential mediators of heavy metal resistance in zebrafish cells

Acquired resistance of mammalian cells to heavy metals is closely relevant to enhanced expression of several multidrug resistance-associated proteins (MRP), but it remains unclear whether MRP proteins confer resistance to heavy metals in zebrafish. In this study, we obtained zebrafish (Danio rerio) fibroblast-like ZF4 cells with resistance to toxic heavy metals after chronic cadmium exposure and selection for 6months. These cadmium-resistant cells (ZF4-Cd) were maintained in 5μM cadmium and displayed cross-resistance to cadmium, mercury, arsenite and arsenate. ZF4-Cd cells remained the resistance to heavy metals after protracted culture in cadmium-free medium. In comparison with ZF4-WT cells, ZF4-Cd cells exhibited accelerated rate of cadmium excretion, enhanced activity of MRP-like transport, elevated expression of abcc2, abcc4 and mt2 genes, and increased content of cellular GSH. Inhibition of MRP-like transport activity, GSH biosynthesis and GST activity significantly attenuated the resistance of ZF4-Cd cells to heavy metals. The results indicate that some of MRP transporters are involved in the efflux of heavy metals conjugated with cellular GSH and thus play crucial roles in heavy metal detoxification of zebrafish cells.     

Cigarette smoke extract affects functional activity of MRP1 in bronchial epithelial cells

Cigarette smoke is the principal risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) superfamily of transporters, which transport physiologic and toxic substrates across cell membranes. MRP1 is highly expressed in lung epithelium. This study aims to analyze the effect of cigarette smoke extract (CSE) on MRP1 activity. In the human bronchial epithelial cell line 16HBE14o-, MRP1 function was studied flow cytometrically by cellular retention of carboxyfluorescein (CF) after CSE incubation and MRP1 downregulation by RNA interference (siRNA). Cell survival was measured by the MTT assay. Immunocytochemically, it was shown that 16HBE14o(-) expressed MRP1 and breast cancer resistance protein. Coincubation of CSE IC50 (1.53% +/- 0.22%) with MK571 further decreased cell survival 31% (p, = 0.018). CSE increased cellular CF retention dose dependently from 1.7-fold at 5% CSE to 10.3-fold at 40% CSE (both p < 0.05). siRNA reduced MRP1 RNA expression with 49% and increased CF accumulation 67% versus control transfected cells. CSE exposure further increased CF retention 24% (p = 0.031). A linear positive relation between MRP1 function and CSE-modulating effects (r = 0.99, p =0.089) was shown in untransfected, control transfected, and MRP1 downregulated 16HBE14o- cells analogous to blocking effects with MRP1 inhibitor MK571 (r = 0.99, p = 0.034). In conclusion, cigarette smoke extract affects MRP1 activity probably competitively in bronchial epithelial cells. Inhibition of MRP1 in turn results in higher CSE toxicity. We propose that MRP1 may be a protective protein for COPD development.     

Transport of cyclic nucleotides and estradiol 17-beta-D-glucuronide by multidrug resistance protein 4. Resistance to 6-mercaptopurine and 6-thioguanine.

Human multidrug resistance protein 4 (MRP4) has recently been determined to confer resistance to the antiviral purine analog 9-(2-phosphonylmethoxyethyl)adenine and methotrexate. However, neither its substrate selectivity nor physiological functions have been determined. Here we report the results of investigations of the in vitro transport properties of MRP4 using membrane vesicles prepared from insect cells infected with MRP4 baculovirus. It is shown that expression of MRP4 is specifically associated with the MgATP-dependent transport of cGMP, cAMP, and estradiol 17-beta-D-glucuronide (E(2)17 beta G). cGMP, cAMP, and E(2)17 beta G are transported with K(m) and V(max) values of 9.7 +/- 2.3 microm and 2.0 +/- 0.3 pmol/mg/min, 44.5 +/- 5.8 microm and 4.1 +/- 0.4 pmol/mg/min, and 30.3 +/- 6.2 microm and 102 +/- 16 pmol/mg/min, respectively. Consistent with its ability to transport cyclic nucleotides, it is demonstrated that the MRP4 drug resistance profile extends to 6-mercaptopurine and 6-thioguanine, two anticancer purine analogs that are converted in the cell to nucleotide analogs. On the basis of its capacity to transport cyclic nucleotides and E(2)17 beta G, it is concluded that MRP4 may influence diverse cellular processes regulated by cAMP and cGMP and that its substrate range is distinct from that of any other characterized MRP family member.     

Effect of bile salts on the biliary excretion of glycodihydrofusidate in the rat

The biliary elimination of glycodihydrofusidate (GDHF), a structural analogue of bile salts, was studied in bile fistula rats. GDHF was excreted in bile with a maximal excretory rate (Tm = 0.80 mumol min-1 kg-1) which is much lower than bile salts Tm. The effects of dehydrocholate and taurocholate on GDHF biliary secretion suggest a stimulatory effect of bile salts on canalicular excretion of the drug. (a) When a bolus intravenous injection of 3 mumol of GDHF was followed after 2 min by a continuous dehydrocholate perfusion (10 mumol min-1 kg-1), biliary excretion of GDHF was increased in comparison with control rats. (b) Upon attaining the biliary Tm by continuous perfusion of GDHF at a rate of 1.35 mumol min-1 kg-1, infusion with either taurocholate or dehydrocholate increased its Tm to a similar degree. These results are similar to those previously obtained with the effects of bile salt infusions on the Tm of bromosulfophthalein. They suggest therefore that hepatic transport of GDHF and bile salts occurs by routes which are distinct for canalicular transport in spite of the striking structural similarities between GDHF and bile salts.     

Taurine supplementation induces multidrug resistance protein 2 and bile salt export pump expression in rats and prevents endotoxin-induced cholestasis

The effect of oral taurine supplementation on endotoxin-induced cholestasis was investigated in rat liver. At 12h following lipopolysaccharide (LPS) injection (4mg/kg body weight i.p.) bile flow and bromosulfophthalein (BSP) and taurocholate (TC) excretion were determined in the perfused liver and the expression of the canalicular transporters multidrug resistance protein 2 (Mrp2) and bile salt export pump (Bsep) was analyzed. Injection of LPS induced a significant decrease of bile flow ( 2.2+/-0.2 microl/g liver wet weight/min vs 3.3+/-0.1 microl/g liver wet weight in controls), biliary BSP excretion (10.8+/-2.2 nmol/g/min vs 21.0+/-3.8 nmol/g/min), and biliary TC excretion (114+/-23 nmol/g/min vs 228+/-8 nmol/g/min). These effects were due to transporter retrieval from the canalicular membrane and downregulation of Mrp2 and Bsep expression. In taurine-supplemented rats bile flow was 30% higher than that in untreated rats and the expression of Mrp2 and Bsep protein was increased two- to threefold. In taurine-supplemented rats there was no significant reduction of bile flow or of BSP and TC excretion at 12h following LPS injection. This protective effect of taurine was due to higher Mrp2 and Bsep protein levels compared to nonsupplemented LPS-treated rats, whereas relative Mrp2 retrieval from the canalicular membrane induced by LPS was not significantly different. LPS-induced tumor necrosis factor alpha and interleukin-1beta release were lower in taurine-fed rats; however, downregulation of Mrp2 and Bsep expression by LPS was delayed but not prevented. The data show that oral supplementation of taurine induces Mrp2 and Bsep expression and may prevent LPS-induced cholestasis.     

The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides

Cellular export of cyclic nucleotides has been observed in various tissues and may represent an elimination pathway for these signaling molecules, in addition to degradation by phosphodiesterases. In the present study we provide evidence that this export is mediated by the multidrug resistance protein isoform MRP5 (gene symbol ABCC5). The transport function of MRP5 was studied in V79 hamster lung fibroblasts transfected with a human MRP5 cDNA. An MRP5-specific antibody detected an overexpression of the glycoprotein of 185 +/- 15 kDa in membranes from MRP5-transfected cells and a low basal expression of hamster Mrp5 in control membranes. ATP-dependent transport of 3',5'-cyclic GMP at a substrate concentration of 1 micrometer was 4-fold higher in membrane vesicles from MRP5-transfected cells than in control membranes. This transport was saturable with a K(m) value of 2.1 micrometer. MRP5-mediated transport was also detected for 3',5'-cyclic AMP at a lower affinity, with a K(m) value of 379 micrometer. A potent inhibition of MRP5-mediated transport was observed by several compounds, known as phosphodiesterase modulators, including trequinsin, with a K(i) of 240 nm, and sildenafil, with a K(i) value of 267 nm. Thus, cyclic nucleotides are physiological substrates for MRP5; moreover, MRP5 may represent a novel pharmacological target for the enhancement of tissue levels of cGMP.