Estradiol-17beta-D-glucuronide induces endocytic internalization of Bsep in rats

Endocytic internalization of the multidrug resistance-associated protein 2 (Mrp2) was previously suggested to be involved in estradiol-17beta-D-glucuronide (E217G)-induced cholestasis. Here we evaluated in the rat whether a similar phenomenon occurs with the bile salt export pump (Bsep) and the ability of DBcAMP to prevent it. E217G (15 micromol/kg i.v.) impaired bile salt (BS) output and induced Bsep internalization, as assessed by confocal microscopy and Western blotting. Neither cholestasis nor Bsep internalization occurred in TR- rats lacking Mrp2. DBcAMP (20 micromol/kg i.v.) partially prevented the decrease in bile flow and BS output and substantially prevented E217G-induced Bsep internalization. In hepatocyte couplets, E217G (50 microM) diminished canalicular accumulation of a fluorescent BS and decreased Bsep-associated fluorescence in the canalicular membrane; DBcAMP (10 microM) fully prevented both effects. In conclusion, our results suggest that changes in Bsep localization are involved in E217G-induced impairment of bile flow and BS transport and that DBcAMP prevents this effect by stimulating insertion of canalicular transporter-containing vesicles. Mrp2 is required for E217G to induce its harmful effect.     

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

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

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.     

Phloracetophenone-induced choleresis in rats is mediated through Mrp2

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

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.     

Retrieval of the mrp2 gene encoded conjugate export pump from the canalicular membrane contributes to cholestasis induced by tert-butyl hydroperoxide and chloro-dinitrobenzene

Oxidative stress is known to induce cholestasis, but the underlying mechanisms are poorly understood. In this study we have characterized the short-term effects of tert-butyl hydroperoxide (t-BOOH)- and 1-chloro-2,4-dinitrobenzene (CDNB) on the mrp2 gene encoded canalicular export pump (Mrp2). The effects of t-BOOH and CDNB on bile formation, tissue GSH levels and subcellular Mrp2 localization were studied in perfused rat liver. Both, t-BOOH (0.5 mM) and CDNB (0.1 mM) induced within 60 min a decrease of hepatic GSH levels by more than 90% and an almost complete cessation of bile flow. As revealed by confocal laser scanning microscopy, this cholestasis was accompanied by a loss of immunoreactive MRP2 from the canalicular membrane and its appearance inside the hepatocytes in putative intracellular vesicles. On the other hand, the intracellular distribution of dipeptidyl peptidase IV (DPPIV), another canalicular protein, and of zonula occludens associated polypeptide (ZO-1) remained unaffected, indicating selectivity of the Mrp2 retrieval pattern. Both, t-BOOH and CDNB induced a rapid net K+ efflux from the liver and a significant decrease of liver cell hydration. We conclude that severe glutathione depletion induces cholestasis by a retrieval of Mrp2, but not of DPPIV from the canalicular membrane. The underlying mechanism is unclear; however, a decrease in liver cell hydration, which occurs under these conditions, may contribute to this effect.     

ERK1/2 and p38 MAPKs Are Complementarily Involved in Estradiol 17?-d-Glucuronide-Induced Cholestasis: Crosstalk with cPKC and PI3K

Objective

The endogenous, cholestatic metabolite estradiol 17ß-d-glucuronide (E₂17G) induces endocytic internalization of the canalicular transporters relevant to bile formation, Bsep and Mrp2. We evaluated here whether MAPKs are involved in this effect.

Design

ERK1/2, JNK1/2, and p38 MAPK activation was assessed by the increase in their phosphorylation status. Hepatocanalicular function was evaluated in isolated rat hepatocyte couplets (IRHCs) by quantifying the apical secretion of fluorescent Bsep and Mrp2 substrates, and in isolated, perfused rat livers (IPRLs), using taurocholate and 2,4-dinitrophenyl-S-glutathione, respectively. Protein kinase participation in E₂17G-induced secretory failure was assessed by co-administering selective inhibitors. Internalization of Bsep/Mrp2 was assessed by confocal microscopy and image analysis.

Results

E₂17G activated all kinds of MAPKs. The PI3K inhibitor wortmannin prevented ERK1/2 activation, whereas the cPKC inhibitor Gö6976 prevented p38 activation, suggesting that ERK1/2 and p38 are downstream of PI3K and cPKC, respectively. The p38 inhibitor SB203580 and the ERK1/2 inhibitor PD98059, but not the JNK1/2 inhibitor SP600125, partially prevented E₂17G-induced changes in transporter activity and localization in IRHCs. p38 and ERK1/2 co-inhibition resulted in additive protection, suggesting complementary involvement of these MAPKs. In IPRLs, E₂17G induced endocytosis of canalicular transporters and a rapid and sustained decrease in bile flow and biliary excretion of Bsep/Mrp2 substrates. p38 inhibition prevented this initial decay, and the internalization of Bsep/Mrp2. Contrarily, ERK1/2 inhibition accelerated the recovery of biliary secretion and the canalicular reinsertion of Bsep/Mrp2.

Conclusions

cPKC/p38 MAPK and PI3K/ERK1/2 signalling pathways participate complementarily in E₂17G-induced cholestasis, through internalization and sustained intracellular retention of canalicular transporters, respectively.     

Canalicular Mrp2 localization is reversibly regulated by the intracellular redox status

Oxidative stress is known to be a common feature of cholestatic syndrome. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile salt-independent bile flow, under acute oxidative stress, and a series of signaling pathways finally leading to the activation of novel protein kinase C were involved in this mechanism; however, it has been unclear whether the internalized Mrp2 localization was relocalized to the canalicular membrane when the intracellular redox status was recovered from oxidative stress. In this study, we demonstrated that decreased canalicular expression of Mrp2 induced by tertiary-butyl hydroperoxide (t-BHP) was recovered to the canalicular membrane by the replenishment of GSH by GSH-ethyl ester, a cell-permeable form of GSH. Moreover, pretreatment of isolated rat hepatocytes with colchicine and PKA inhibitor did not affect the t-BHP-induced Mrp2 internalization process but did prevent the Mrp2 recycling process induced by GSH replenishment. Moreover, intracellular cAMP concentration similarly changed with the change of intracellular GSH content. Taken together, our data clearly indicate that the redox-sensitive balance of PKA/PKC activation regulates the reversible Mrp2 localization in two different pathways, the microtubule-independent internalization pathway and -dependent recycling pathway of Mrp2.     

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.     

The effect of colchicine on the development of lithocholic acid-induced cholestasis. A study of the role of microtubules in intracellular cholesterol transport.

The pathogenesis of lithocholic acid (LCA-Na)-induced cholestasis involves a rapid accumulation of cholesterol in the bile canalicular membrane. Since microtubules play an important role in the intracellular transport of many materials, including cholesterol, the present study was undertaken to assess the extent to which they participate in the development of LCA-Na-induced cholestasis. Rats were pretreated with either colchicine (0.2 mumol/100 g body wt.) or saline solution 90 min before injection with LCA-Na (12 mumol/100 g body wt.). Colchicine, although not increasing bile flow by itself, significantly reduced the cholestasis caused by LCA-Na (57-32% reduction in bile flow) without affecting its metabolism into less toxic bile acids or its distribution in blood, liver or bile. Bile canalicular membranes isolated from animals treated with a combination of colchicine and LCA-Na contained less cholesterol than those treated with LCA-Na alone. However, membranes obtained from rats treated with colchicine alone contained much less cholesterol than did controls. It was found that the total amount of cholesterol accumulated within the bile canalicular membrane following LCA-Na treatment (LCA-Na + colchicine versus colchicine alone compared with LCA-Na versus controls) was unchanged by colchicine treatment. In view of these findings it is suggested that the total amount of cholesterol present within the bile canalicular membrane determines the extent of LCA-Na-induced cholestasis, LCA-Na probably moves cholesterol to the bile canalicular membrane via a microtubule independent pathway, and microtubules are unlikely to function in the transcellular transport of LCA-Na.     

Oxidative stress is a triggering factor for LPS-induced Mrp2 internalization in the cryopreserved rat and human liver slices

Cholestasis develops during inflammation and is characterized as occurring under oxidative stress. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid or lipopolysaccharide (LPS)-induced acute oxidative stress in rat liver. However, it remains unclear whether canalicular Mrp2 internalization is observed in human liver under conditions of acute oxidative stress. In this study, we examined the effect of dimerumic acid (DMA), an antioxidant and found in traditional Chinese medicine, on endotoxin-induced Mrp2 internalization in rat and human liver slices. At 1.5 h following LPS treatment (100 μg/mL), canalicular Mrp2 localization was disrupted without changing the expression of Mrp2 protein or the integrity of filamentous actin in the rat and human liver slices. Pretreatment with DMA (10 μM) counteracted LPS-induced subcellular distribution of Mrp2. Our data clearly indicated that LPS-induced short-term rapid retrieval of Mrp2 from the canalicular surface resulted from LPS-induced oxidative stress in rat and human liver slices.     

The metabolism and excretion of oestradiol-3 beta-D-glucuronide in rats

1. Tritium labelled oestradiol-3 beta-D-glucuronide (E2-3G) was synthesised by sodium borohydride reduction of labelled oestrone-glucuronide (E1-G) and injected intravenously into anaesthetised rats. Bile and urine were collected to assess the routes and rate of excretion of E2-3G. Bile and urine samples were analysed by reverse phase HPLC to determine the metabolites of E2-3G. 2. When E2-3G was given at 11 and 22 mumol/kg, 83 and 85% respectively was excreted in bile within 3 hr and 1 and 3% in urine. 3. The major metabolite was E1-G which accounted for 89 and 92% respectively of the injected E2-3G which was recovered in bile. 4. It is concluded that bile is the major route of excretion of E2-3G in rats and that it is converted mainly to E1-G before excretion.     

Ca2+-dependent protein kinase C isoforms induce cholestasis in rat liver

Bile secretion is regulated by different signaling transduction pathways including protein kinase C (PKC). However, the role of different PKC isoforms for bile formation is still controversial. This study investigates the effects of PKC isoform selective activators and inhibitors on PKC translocation, bile secretion, bile acid uptake, and subcellular transporter localization in rat liver, isolated rat hepatocytes and in HepG2 cells. In rat liver activation of Ca(2+)-dependent cPKCalpha and Ca(2+)-independent PKCepsilon by phorbol 12-myristate 13-acetate (PMA, 10nmol/liter) is associated with their translocation to the plasma membrane. PMA also induced translocation of the cloned rat PKCepsilon fused to a yellow fluorescent protein (YFP), which was transfected into HepG2 cells. In the perfused liver, PMA induced marked cholestasis. The PKC inhibitors G?6850 (1 micromol/liter) and G?6976 (0.2 micromol/liter), a selective inhibitor of Ca(2+)-dependent PKC isoforms, diminished the PMA effect by 50 and 60%, respectively. Thymeleatoxin (Ttx,) a selective activator of Ca(2+)-dependent cPKCs, did not translocate rat PKCepsilon-YFP transfected in HepG2 cells. However, Ttx (0.5-10 nmol/liter) induced cholestasis similar to PMA and led to a retrieval of Bsep from the canalicular membrane in rat liver while taurocholate-uptake in isolated hepatocytes was not affected. G?6976 completely blocked the cholestatic effect of Ttx but had no effect on tauroursodeoxycholate-induced choleresis. The data identify Ca(2+)-dependent PKC isoforms as inducers of cholestasis. This is mainly due to inhibition of taurocholate excretion involving transporter retrieval from the canalicular membrane.     

ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (Mrp3)

We have previously shown that cloned rat multidrug resistance-associated protein 3 (Mrp3) has the ability to transport organic anions such as 17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG) and has a different substrate specificity from MRP1 and MRP2 in that glutathione conjugates are poor substrates for Mrp3 (Hirohashi, T., Suzuki, H., and Sugiyama, Y. (1999) J. Biol. Chem. 274, 15181-15185). In the present study, the involvement of Mrp3 in the transport of endogenous bile salts was investigated using membrane vesicles from LLC-PK1 cells transfected with rat Mrp3 cDNA. The ATP-dependent uptake of [(3)H]taurocholate (TC), [(14)C]glycocholate (GC), [(3)H]taurochenodeoxycholate-3-sulfate (TCDC-S), and [(3)H]taurolithocholate-3-sulfate (TLC-S) was markedly stimulated by Mrp3 transfection in LLC-PK1 cells. The extent of Mrp3-mediated transport of bile salts was in the order, TLC-S > TCDC-S > TC > GC. The K(m) and V(max) values for the uptake of TC and TLC-S were K(m) = 15.9 +/- 4.9 microM and V(max) = 50.1 +/- 9.3 pmol/min/mg of protein and K(m) = 3.06 +/- 0.57 microM and V(max) = 161.9 +/- 21.7 pmol/min/mg of protein, respectively. At 55 nM [(3)H]E(2)17betaG and 1.2 microM [(3)H]TC, the apparent K(m) values for ATP were 1.36 and 0.66 mM, respectively. TC, GC, and TCDC-S inhibited the transport of [(3)H]E(2)17betaG and [(3)H]TC to the same extent with an apparent IC(50) of approximately 10 microM. TLC-S inhibited the uptake of [(3)H]E(2)17betaG and [(3)H]TC most potently (IC(50) of approximately 1 microM) among the bile salts examined, whereas cholate weakly inhibited the uptake (IC(50) approximately 75 microM). Although TC and GC are transported by bile salt export pump/sister of P-glycoprotein, but not by MRP2, and TCDC-S and TLC-S are transported by MRP2, but not by bile salt export pump/sister of P-glycoprotein, it was found that Mrp3 accepts all these bile salts as substrates. This information, together with the finding that MRP3 is extensively expressed on the basolateral membrane of human cholangiocytes, suggests that MRP3/Mrp3 plays a significant role in the cholehepatic circulation of bile salts.     

Involvement of oxidative stress in the impairment in biliary secretory function induced by intraperitoneal administration of aluminum to rats

We have shown that aluminum (Al) induces cholestasis associated with multiple alterations in hepatocellular transporters involved in bile secretory function, like Mrp2. This work aims to investigate whether these harmful effects are mediated by the oxidative stress caused by the metal. For this purpose, the capability of the antioxidant agent, vitamin E, to counteract these alterations was studied in male Wistar rats. Aluminum hydroxide (or saline in controls) was administered ip (27 mg/kg body weight, three times a week, for 90 d). Vitamin E (600 mg/kg body weight) was coadministered, sc. Al increased lipid peroxidation (+50%) and decreased hepatic glutation levels (-43%) and the activity of glutation peroxidase (-50%) and catalase (-88%). Vitamin E counteracted these effects total or partially. Both plasma and hepatic Al levels reached at the end of the treatment were significantly reduced by vitamin E (-40% and -44%, respectively;p< 0.05). Al increased 4 times the hepatic apoptotic index, and this effect was fully counteracted by vitamin E. Bile flow was decreased in Al-treated rats (-37%) and restored to normality by vitamin E. The antioxidant normalized the hepatic handling of the Mrp2 substrates, rose bengal, and dinitrophenyl-S-glutathione, which was causally associated with restoration of Mrp2 expression. Our data indicate that oxidative stress has a crucial role in cholestasis, apoptotic/necrotic hepatocellular damage, and the impairment in liver transport function induced by Al and that vitamin E counteracts these harmful effects not only by preventing free-radical formation but also by favoring Al disposal.     

Bile acid secretion and pool size during phenobarbital induced hypercholeresis

An increase in bile flow after phenobarbital administration occurs in the rat and other species; however, the mechanism(s) of the choleretic effect is incompletely understood and the role of the increase in liver weight is controversial. We therefore measured bile flow, bile acid secretion and pool size in male Sprague-Dawley rats pretreated with phenobarbital (75 mg/kg/day) for 6 days; liver weight, liver cell volume and DNA content were also evaluated. Phenobarbital treatment increased liver weight and mean hepatocyte volume by 39 and 26%, respectively, while total DNA content did not change, thus indicating that the hepatomegaly results principally from hypertrophy rather than hyperplasia. Bile flow was significantly higher in treated rats when expressed per unit of body weight (64.6 +/- 2.4 (S.E.) vs 53.3 +/- 1.6 microliter/min/kg; P less than 0.05) but was unchanged when expressed per gram of liver (1.40 +/- 0.04 vs 1.37 +/- 0.06 microliter/min/g; P greater than 0.5). The initial bile acid secretion rate and pool size were both significantly reduced in the phenobarbital group compared to controls (1224.2 +/- 110.4 vs 1656.6 +/- 163.2 nmol/kg/min and 562.8 +/- 41.5 vs 814.3 +/- 78.3 mumol/kg; both P less than 0.05), whereas the basal synthetic rate was unchanged. These findings suggest that the enlarged, phenobarbital-treated hepatocyte produces more bile than the normal cell, despite the decreased secretion of bile acids. Therefore, the drug-induced choleresis involves a selective increase in the bile acid-independent fraction of bile flow.     

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.