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Effect of ursodeoxycholate-3,7-disulfate on biliary excretion of lithocholate-3-O-glucuronide in Eisai hyperbilirubinemic rat (EHBR)
Hepatology Research 23 (2002) 296– 300
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Effect of ursodeoxycholate-3,7-disulfate on biliary excretion of lithocholate-3-O-glucuronide in Eisai hyperbilirubinemic rat (EHBR) Yasumasa Hasegawa, Hajime Takikawa * Department of Medicine, Teikyo Uni6ersity School of Medicine, Itabashi-ku, Tokyo 173 -8605, Japan Received 20 September 2001; received in revised form 21 November 2001; accepted 5 December 2001
Abstract Biliary excretion of lithocholate-3-O-glucuronide and ursodeoxycholate-3,7-disulfate is markedly impaired in EHBR, in which the multidrug resistance protein 2 (Mrp2), a major ATP-dependent canalicular organic anion transporter, is impaired. We previously reported that biliary excretion of lithocholate-3-sulfate was enhanced by ursodeoxycholate-3,7-disulfate in Eisai hyperbilirubinemic rat (EHBR). In the present study, we examined the effect of ursodeoxycholate-3,7-disulfate infusion on biliary excretion of lithocholate-3-O-glucuronide. Although in control rats, ursodeoxycholate-3,7-disulfate infusion had no effect on biliary lithocholate-3-O-glucuronide excretion, in EHBR it enhanced biliary lithocholate-3-O-glucuronide excretion. These findings indicate that ursodeoxycholate-3,7disulfate may interact to the excretory pathway of lithocholate-3-O-glucuronide and enhances biliary excretion of lithocholate-3-O-glucuronide in EHBR. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Lithocholate-3-O-glucuronide; Ursodeoxycholate-3,7-disulfate; Taurocholate-3-sulfate; Bile acids; Biliary excretion; Eisai hyperbilirubinemic rats (EHBR); Multidrug resistance protein 2 (Mrp2)
1. Introduction Eisai hyperbilirubinemic rat (EHBR) is a mutant Sprague –Dawley rat (SDR) with conjugated hyperbilirubinemia due to a genetic defect of a canalicular ATP-dependent transporter, multidrug resistance protein 2 (Mrp2) [1 – 4]. Biliary
* Corresponding author. Tel.: +81-3-3964-1211; fax: + 813-3964-8477 E-mail address: [email protected] (H. Takikawa).
excretion of organic anions and bile acid sulfates and glucuronides has been reported to be markedly impaired in EHBR [1,2,5 –21]. In EHBR, although the biliary excretion of lithocholate-3-sulfate (3-sul-LC) and lithocholate3-O-glucuronide (3-glcA-LC) is markedly delayed, their excretion still occurs [1]. 3-Sul-LC is excreted into bile mainly as a taurine conjugate (3-sul-LC-tau) both in EHBR and SDR [1]. Recently, Akita et al. reported that ATP-dependent 3-sul-LC-tau transport was defective in canalicular membrane vesicles of EHBR, and that its transport was observed in Mrp2-transfected cells
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Y. Hasegawa, H. Takikawa / Hepatology Research 23 (2002) 296–300
[22]. These data gave direct evidences that 3-sulLC-tau was a substrate for Mrp2. Ursodeoxycholate-3,7-disulfate (3,7-sul-UDC) acts as an organic anion, which caused the uncoupling of bile acid and lipid excretion [5], and its biliary excretion is markedly delayed in EHBR [7]. 3,7-Sul-UDC is excreted into bile without any further metabolism [5]. However, its excretory mechanism is unknown. We previously reported that 3,7-sul-UDC infusion enhanced biliary 3-sul-LC excretion in EHBR, whereas such findings were not observed in SDR [14]. Therefore, it is interesting to study whether a similar enhancement of biliary excretion by 3,7-sul-UDC is observed with other bile acid conjugates, 3-glcA-LC and taurocholate-3sulfate (3-sul-C-tau), which are more hydrophilic than 3-sul-LC and are also considered to be substrates for Mrp2 [17]. In the present study, the effect of 3,7-sulUDC infusion on biliary 3-glcA-LC excretion was examined in EHBR and SDR. Furthermore, the effect of the infusion of dibromosulfophthalein (DBSP), an Mrp2 substrate [9,23], on biliary 3-glcA-LC excretion, and the effect of 3,7-sul-UDC infusion on biliary 3-sul-C-tau excretion was also examined in EHBR
2. Materials and methods
2.1. Materials 3,7-Sul-UDC (98% pure) was kindly supplied by Mitsubishi-Tokyo Pharmaceuticals, Inc. (Tokyo, Japan). [14C]3-GlcA-LC was synthesized by the glucuronidation of [14C]lithocholate (1990 MBq/mmol, Buckinghamshire, UK), and [14C]3sul-C-tau was obtained by the conjugation with [14C]taurine (4015 MBq/mmol, DuPont/NEN, Boston, MA, USA) of cholate-3-sulfate, as reported previously [1,17]. Male SDRs were purchased from Japan Laboratory Animals Inc. (Saitama, Japan), and EHBRs were obtained from Sankyo Labo Service Co. (Tokyo, Japan).
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2.2. Experimental procedures Male EHBR and SDR (270 g) were used after overnight fasting. The rats were anesthetized with an intraperitoneal injection of pentobarbital (5 mg/100 g b.wt.), and the common bile duct was cannulated with a PE-10 tube (Beckon Dickinson Primary Care Diagnostics, Franklin Lake, NJ). The femoral vein was cannulated with a 3-Fr venous catheter, and 3% human albumin in 5% glucose solution (standard solution) was infused at the rate of 2 ml/h during the experiment. Sixty minutes after bile duct cannulation, tracer doses of bile acid conjugates dissolved in 50 ml of the standard solution was injected via the femoral vein. Bile samples were collected every 10 min for 60 min and counted for radioactivity. 1. A tracer dose (2.2 kBq) of [14C]3-glcA-LC was intravenously injected to SDR and EHBR. 3,7-Sul-UDC infusion (0.2 mmol/min per 100 g b.wt.) was started 30 min before 3-glcA-LC injection and was continued until the end of the experiments. 2. A tracer dose (2.2 kBq) of [14C]3-glcA-LC was intravenously injected to EHBR. DBSP infusion (0.2 mmol/min per 100 g b.wt.) was started 30 min before 3-glcA-LC injection and was continued until the end of the experiments. 3. A tracer dose (0.8 kBq) of [14C]3-sul-C-tau was intravenously injected to EHBR, and 3,7-sulUDC infusion (0.2 mmol/min per 100 g b.wt.) was started 30 min before 3-sul-C-tau injection and was continued until the end of the experiments.
2.3. Statistical analysis All data of biliary excretion were expressed as means9S.D. of percentages of total administered radioactivity per 10 min. A statistical analysis was performed for the data of cumulative biliary excretion in 60 min by the Mann– Whitney U-test, and PB 0.05 was considered to indicate a significant difference.
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3. Results Although 3,7-sul-UDC infusion increased bile flow, it had no effect on biliary 3-glcA-LC excretion in SDR (Fig. 1); biliary excretion of 3-glcALC in 60 min was 8496.8 and 879 4.9% in the presence and absence of 3,7-sul-UDC infusion, respectively. In contrast, biliary 3-glcA-LC excretion was markedly enhanced by 3,7-sul-UDC infusion in EHBR (Fig. 2); biliary excretion of 3-glcA-LC in 60 min was 239 4.2 and 139 1.5% in the presence and absence of 3,7-sul-UDC infusion, respectively (P B0.05). Biliary 3-glcA-LC excretion was impaired in EHBR as compared with SDR (Figs. 1 and 2), as reported previously [1]. DBSP infusion decreased biliary 3-glcA-LC excretion in EHBR (Fig. 3), biliary excretion of 3-glcA-LC in 60 min was 6.49 2.4% in the presence of DBSP infusion (P B0.05 vs. control). 3,7-Sul-UDC infusion had no effect on biliary 3-sul-C-tau excretion in EHBR (Fig. 4); biliary
Fig. 2. Effect of 3,7-sul-UDC infusion on bile flow and biliary [14C]3-glcA-LC excretion in EHBR. Data are means 9 S.D. of five control experiments (open circles) and three experiments of 3,7-sul-UDC infusion at the rate of 0.2 mmol/min per 100 g b. wt. started 30 min before 3-glcA-LC injection (closed circles).
excretion of 3-sul-C-tau in 60 min was 789 9.5 and 89 913% in the presence and absence of 3,7-sul-UDC infusion, respectively. 4. Discussion
Fig. 1. Effect of 3,7-sul-UDC infusion on bile flow and biliary [14C]3-glcA-LC excretion in SDR. Data are means 9 S.D. of five control experiments (open circles) and three experiments of 3,7-sul-UDC infusion at the rate of 0.2 mmol/min per 100 g b. wt. started 30 min before 3-glcA-LC injection (closed circles).
Although biliary excretion of both 3-glcA-LC and 3,7-sul-UDC was markedly impaired in EHBR [1,7], the infusion of 3,7-sul-UDC had no effect on biliary 3-glcA-LC excretion in SDR (Fig. 1). These data suggest that biliary excretory pathways of 3-glcA-LC and 3,7-sul-UDC are different, since the infusion rate of 3,7-sul-UDC in the present study (0.2 mmol/min per 100 g b.wt.) was above its excretory maximum, which is estimated to be approximately 0.1 mmol/min per 100 g b.wt. [24]. Although a defect of Mrp2 was reported in EHBR [3,4], the results of the present study as well as previous studies [20,21,25,26] suggest that the impaired biliary excretion of some organic anions and bile acid conjugates is due to the secondary effect of the Mrp2 defect.
Y. Hasegawa, H. Takikawa / Hepatology Research 23 (2002) 296–300
The enhanced biliary excretion of 3-glcA-LC by 3,7-sul-UDC observed in EHBR (Fig. 2) is considered to be due to 1) the displacement of 3-glcALC bound to albumin, thus increasing its uptake by the liver, 2) enhanced sinusoidal uptake of 3-glcA-LC, 3) the displacement of 3-glcA-LC bound to cytosolic binders, which are considered to be important for intrahepatic transport, and 4) the interaction of canalicular transporters for 3glcA-LC and 3,7-sul-UDC. We previously reported a similar enhancement of biliary 3-sul-LC excretion by 3,7-sul-UDC and an absence of an effect by 3,7-sul-UDC on serum clearance of 3sul-LC in EHBR [14]. Unfortunately, the amount of [14C]3-glcA-LC was not enough to study its serum clearance in the present study. However, since the canalicular transport is considered to be a rate limiting step of hepatobiliary transport of organic anions, an enhancement of the affinity for 3-glcA-LC of a canalicular transporter which is present in EHBR, an Mrp2-defective rat, due to a conformational change of the transporter is the
Fig. 3. Effect of DBSP infusion on bile flow and biliary [14C]3-glcA-LC excretion in EHBR. Data are means 9 S.D. of five control experiments (open circles) and three experiments of 3,7-sul-UDC infusion at the rate of 0.2 mmol/min per 100 g b. wt. started 30 min before 3-glcA-LC injection (closed circles).
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Fig. 4. Effect of 3,7-sul-UDC infusion on bile flow and biliary [14C]3-sul-C-tau excretion in EHBR. Data are means 9 S.D. of five control experiments (open circles) and three experiments of 3,7-sul-UDC infusion at the rate of 0.2 mmol/min per 100 g b. wt. started 30 min before 3-sul-C-tau injection (closed circles).
most likely mechanism of the enhanced 3-glcALC excretion by 3,7-sul-UDC. A report by Niinuma et al. that E3040-sulfate enhanced the transport of S-(2,4-dinitrophenyl)-glutathione by canalicular membrane vesicles of SDR [25] further supports such possibility of a conformational change of an unknown transporter. In contrast to the results with 3,7-sul-UDC, DBSP inhibited biliary 3-glcA-LC excretion in EHBR (Fig. 3). We previously reported that biliary excretion of DBSP was markedly reduced in EHBR and that DBSP inhibited biliary 3-sul-LC excretion both in SDR and EHBR [9]. Accordingly, DBSP is considered to compete to canalicular 3-glcA-LC transport by unknown transporter(s), which is present in EHBR. In contrast to the results with 3-glcA-LC (Fig. 2) and 3-sul-LC [14], biliary 3-sul-C-tau excretion was not affected by 3,7-sul-UDC in EHBR (Fig. 4). We previously reported that the extent of the impairment of biliary excretion of 3-sul-C-tau was not so prominent as that of 3-sul-LC-tau [17] and
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3-glcA-LC [1], suggesting that the involvement of Mrp2 on canalicular 3-sul-C-tau excretion is smaller than that of 3-sul-LC-tau and 3-glcA- LC. The absence of the effect of 3,7-sul-UDC on 3-sul-C-tau excretion in EHBR suggests a complete difference of the canalicular transport of these hydrophilic bile acid conjugates in the absence of Mrp2. In conclusion, 3,7-sul-UDC may interact to the excretory pathway of 3-glcA-LC, possibly by a confirmational change of an unknown canalicular transporter, and enhances biliary excretion of 3glcA-LC in EHBR.
References [1] Takikawa H, Sano N, Narita T, et al. Biliary excretion of bile acid conjugates in a hyperbilirubinemic mutant Sprague – Dawley rat. Hepatology 1991;14:352 –60. [2] Hosokawa S, Tagaya O, Mikami T, et al. A new rat mutant with chronic conjugated hyperbilirubinemia and renal glomerular lesions. Lab Anim Sci 1991;42:27 – 34. [3] Ito K, Suzuki H, Hirohashi T, Kume K, Shimizu T, Sugiyama Y. Expression of a putative ATP-binding cassette region, homologous to that in multidrug resistance-associated protein (MRP), is hereditarily defective in Eisai hyperbilirubinemic rats (EHBR). Int Hepatol Commun 1996;4:291 – 8. [4] Ito K, Suzuki H, Hirohashi T, Kume K, Shimizu T, Sugiyama Y. Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR. Am J Physiol 1997;272:G16 –22. [5] Takikawa H, Sano N, Minagawa K, Yamanaka M. Effect of ursodeoxycholate, its glucuronide and disulfate and b-muricholate on biliary bicarbonate concentration and biliary lipid excretion. J Hepatol 1992;15:77 –84. [6] Sano N, Takikawa H, Yamanaka M. Estradiol-17-glucuronide-induced cholestasis. Effects of ursodeoxycholate3-O-glucuronide and 3,7-disulfate. J Hepatol 1993;17:241 – 6. [7] Takikawa H, Sano N, Wako Y, Yamanaka M. Effects of organic anions and bile acids on biliary lipid excretion in hyperbilirubinemic mutant Sprague –Dawley rats. J Hepatol 1993;17:247 – 52. [8] Takikawa H, Minagawa K, Sano N, Yamanaka M. Lithocholate-3-O-glucuronide-induced cholestasis. A study with congenital hyperbilirubinemic rats and effects of ursodeoxycholate conjugates. Dig Dis Sci 1993;38:1543 – 8. [9] Takikawa H, Nishikawa K, Sano N, Yamanaka M, Horie T. Mechanisms of biliary excretion of lithocholate-3-sulfate in Eisai hyperbilirubinemic rats (EHBR). Dig Dis Sci 1995;40:1792 – 7. [10] Takikawa H, Sano N, Tadokoro K, Yamanaka M. Biliary excretion of estrone metabolites in the rat. Int Hepatol Commun 1996;5:251 –8.
[11] Takikawa H, Sano N, Uegaki S, Yamanaka M. Effects of bile acid conjugates on biliary excretion of estradiol-17-glucuronide in the rat. Hepatol Res 1997;7:28 – 34. [12] Kitaura K, Takikawa H, Yamanaka M. Effects of organic anions and bile acid conjugates on biliary excretion of LTC4 in the rat. Prostaglandins 1997;54:745 – 55. [13] Fukumura S, Takikawa H, Yamanaka M. Effects of organic anions and bile acid conjugates on biliary excretion of pravastatin in the rat. Pharm Res 1998;15:72 – 6. [14] Takikawa H, Sano N, Ogasawara T, Yamanaka M. Enhanced biliary excretion of lithocholate-3-sulfate by ursodeoxycholate-3,7-disulfate in Eisai hyperbilirubinemic rat (EHBR). Dig Dis Sci 1998;43:188 – 92. [15] Takikawa H, Sano N, Akimoto K, Yamanaka M. Biliary excretion of estrone sulfate in the rat. Hepatol Res 1998;11:201 – 7. [16] Takikawa H, Sano N, Suzuki C, Takada Y, Yamanaka M. Effect of organic anions and taurolithocholate-3-sulfate on biliary excretion of temocapril in the rat. Hepatol Res 1999;15:157 – 62. [17] Uegaki S, Takikawa H, Yamanaka M. Effect of organic anions and bile acid conjugates on biliary excretion of taurine-conjugated bile acid sulfates in the rat. Steroids 1999;64:790 – 5. [18] Ogasawara T, Takikawa H. Biliary excretion of phenolphthalein glucuronide in the rat. Hepatol Res 2001;20:221 – 31. [19] Sathirakul K, Suzuki H, Yasuda K, et al. Kinetic analysis of hepatobiliary transport of organic anions in Eisai hyperbilirubinemic mutant rats (EHBR). J Pharmacol Exp Ther 1993;265:1301 – 12. [20] Shimamura H, Suzuki H, Hanano M, et al. Multiple systems for the biliary excretion of organic anions in rats: liquiritigenin conjugates as model compounds. J Pharmacol Exp Ther 1994;271:370 – 8. [21] Takenaka O, Horie T, Suzuki H, Sugiyama Y. Different biliary excretion systems for glucuronide and sulfate of a model compound; study using Eisai hyperbilirubinemic rats. J Pharmacol Exp Ther 1995;274:1362 – 9. [22] Akita H, Suzuki H, Kinoshita S, Sato N, Takikawa H, Sugiyama Y. Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump. Biochim Biophys Acta 2001;1511:7 – 16. [23] Jansen PLM, Groothuis GMM, Peters WHM, Meijer DFM. Selective hepatobiliary transport defect for organic anions and neutral steroids in mutant rats with hereditary hyperbilirubinemia. Hepatology 1987;7:71 – 6. [24] Takikawa H, Sano N, Yamanaka M. Effects of ursodeoxycholate and its conjugates on biliary glutathione excretion in rats. Dig Dis Sci 1996;41:1953 – 8. [25] Niinuma K, Takenaka O, Horie T, et al. Kinetic analysis of the primary active transport of conjugated metabolites across the bile canalicular membrane: comparative study of S-(2,4-dinitrophenyl)-glutathione and 6-hydroxy-5,7dimethyl - 2 - methylamino - 4 - (3 - pyridylmethyl)benzothiazole glucuronide. J Pharmacol Exp Ther 1997;282:866 – 72. [26] Nishino A, Kato Y, Igarashi T, Sugiyama Y. Both cMOAT/ MRP2 and another unknown transporter(s) are responsible for the biliary excretion of glucuronide conjugate of the nonpeptide angiotensin II antagonist, telmisaltan. Drug Metab Dispos 2000;28:1146 – 8.
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Effect of ursodeoxycholate-3,7-disulfate on biliary excretion of lithocholate-3-O-glucuronide in Eisai hyperbilirubinemic rat (EHBR) Yasumasa Hasegawa, Hajime Takikawa * Department of Medicine, Teikyo Uni6ersity School of Medicine, Itabashi-ku, Tokyo 173 -8605, Japan Received 20 September 2001; received in revised form 21 November 2001; accepted 5 December 2001
Abstract Biliary excretion of lithocholate-3-O-glucuronide and ursodeoxycholate-3,7-disulfate is markedly impaired in EHBR, in which the multidrug resistance protein 2 (Mrp2), a major ATP-dependent canalicular organic anion transporter, is impaired. We previously reported that biliary excretion of lithocholate-3-sulfate was enhanced by ursodeoxycholate-3,7-disulfate in Eisai hyperbilirubinemic rat (EHBR). In the present study, we examined the effect of ursodeoxycholate-3,7-disulfate infusion on biliary excretion of lithocholate-3-O-glucuronide. Although in control rats, ursodeoxycholate-3,7-disulfate infusion had no effect on biliary lithocholate-3-O-glucuronide excretion, in EHBR it enhanced biliary lithocholate-3-O-glucuronide excretion. These findings indicate that ursodeoxycholate-3,7disulfate may interact to the excretory pathway of lithocholate-3-O-glucuronide and enhances biliary excretion of lithocholate-3-O-glucuronide in EHBR. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Lithocholate-3-O-glucuronide; Ursodeoxycholate-3,7-disulfate; Taurocholate-3-sulfate; Bile acids; Biliary excretion; Eisai hyperbilirubinemic rats (EHBR); Multidrug resistance protein 2 (Mrp2)
1. Introduction Eisai hyperbilirubinemic rat (EHBR) is a mutant Sprague –Dawley rat (SDR) with conjugated hyperbilirubinemia due to a genetic defect of a canalicular ATP-dependent transporter, multidrug resistance protein 2 (Mrp2) [1 – 4]. Biliary
* Corresponding author. Tel.: +81-3-3964-1211; fax: + 813-3964-8477 E-mail address: [email protected] (H. Takikawa).
excretion of organic anions and bile acid sulfates and glucuronides has been reported to be markedly impaired in EHBR [1,2,5 –21]. In EHBR, although the biliary excretion of lithocholate-3-sulfate (3-sul-LC) and lithocholate3-O-glucuronide (3-glcA-LC) is markedly delayed, their excretion still occurs [1]. 3-Sul-LC is excreted into bile mainly as a taurine conjugate (3-sul-LC-tau) both in EHBR and SDR [1]. Recently, Akita et al. reported that ATP-dependent 3-sul-LC-tau transport was defective in canalicular membrane vesicles of EHBR, and that its transport was observed in Mrp2-transfected cells
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Y. Hasegawa, H. Takikawa / Hepatology Research 23 (2002) 296–300
[22]. These data gave direct evidences that 3-sulLC-tau was a substrate for Mrp2. Ursodeoxycholate-3,7-disulfate (3,7-sul-UDC) acts as an organic anion, which caused the uncoupling of bile acid and lipid excretion [5], and its biliary excretion is markedly delayed in EHBR [7]. 3,7-Sul-UDC is excreted into bile without any further metabolism [5]. However, its excretory mechanism is unknown. We previously reported that 3,7-sul-UDC infusion enhanced biliary 3-sul-LC excretion in EHBR, whereas such findings were not observed in SDR [14]. Therefore, it is interesting to study whether a similar enhancement of biliary excretion by 3,7-sul-UDC is observed with other bile acid conjugates, 3-glcA-LC and taurocholate-3sulfate (3-sul-C-tau), which are more hydrophilic than 3-sul-LC and are also considered to be substrates for Mrp2 [17]. In the present study, the effect of 3,7-sulUDC infusion on biliary 3-glcA-LC excretion was examined in EHBR and SDR. Furthermore, the effect of the infusion of dibromosulfophthalein (DBSP), an Mrp2 substrate [9,23], on biliary 3-glcA-LC excretion, and the effect of 3,7-sul-UDC infusion on biliary 3-sul-C-tau excretion was also examined in EHBR
2. Materials and methods
2.1. Materials 3,7-Sul-UDC (98% pure) was kindly supplied by Mitsubishi-Tokyo Pharmaceuticals, Inc. (Tokyo, Japan). [14C]3-GlcA-LC was synthesized by the glucuronidation of [14C]lithocholate (1990 MBq/mmol, Buckinghamshire, UK), and [14C]3sul-C-tau was obtained by the conjugation with [14C]taurine (4015 MBq/mmol, DuPont/NEN, Boston, MA, USA) of cholate-3-sulfate, as reported previously [1,17]. Male SDRs were purchased from Japan Laboratory Animals Inc. (Saitama, Japan), and EHBRs were obtained from Sankyo Labo Service Co. (Tokyo, Japan).
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2.2. Experimental procedures Male EHBR and SDR (270 g) were used after overnight fasting. The rats were anesthetized with an intraperitoneal injection of pentobarbital (5 mg/100 g b.wt.), and the common bile duct was cannulated with a PE-10 tube (Beckon Dickinson Primary Care Diagnostics, Franklin Lake, NJ). The femoral vein was cannulated with a 3-Fr venous catheter, and 3% human albumin in 5% glucose solution (standard solution) was infused at the rate of 2 ml/h during the experiment. Sixty minutes after bile duct cannulation, tracer doses of bile acid conjugates dissolved in 50 ml of the standard solution was injected via the femoral vein. Bile samples were collected every 10 min for 60 min and counted for radioactivity. 1. A tracer dose (2.2 kBq) of [14C]3-glcA-LC was intravenously injected to SDR and EHBR. 3,7-Sul-UDC infusion (0.2 mmol/min per 100 g b.wt.) was started 30 min before 3-glcA-LC injection and was continued until the end of the experiments. 2. A tracer dose (2.2 kBq) of [14C]3-glcA-LC was intravenously injected to EHBR. DBSP infusion (0.2 mmol/min per 100 g b.wt.) was started 30 min before 3-glcA-LC injection and was continued until the end of the experiments. 3. A tracer dose (0.8 kBq) of [14C]3-sul-C-tau was intravenously injected to EHBR, and 3,7-sulUDC infusion (0.2 mmol/min per 100 g b.wt.) was started 30 min before 3-sul-C-tau injection and was continued until the end of the experiments.
2.3. Statistical analysis All data of biliary excretion were expressed as means9S.D. of percentages of total administered radioactivity per 10 min. A statistical analysis was performed for the data of cumulative biliary excretion in 60 min by the Mann– Whitney U-test, and PB 0.05 was considered to indicate a significant difference.
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3. Results Although 3,7-sul-UDC infusion increased bile flow, it had no effect on biliary 3-glcA-LC excretion in SDR (Fig. 1); biliary excretion of 3-glcALC in 60 min was 8496.8 and 879 4.9% in the presence and absence of 3,7-sul-UDC infusion, respectively. In contrast, biliary 3-glcA-LC excretion was markedly enhanced by 3,7-sul-UDC infusion in EHBR (Fig. 2); biliary excretion of 3-glcA-LC in 60 min was 239 4.2 and 139 1.5% in the presence and absence of 3,7-sul-UDC infusion, respectively (P B0.05). Biliary 3-glcA-LC excretion was impaired in EHBR as compared with SDR (Figs. 1 and 2), as reported previously [1]. DBSP infusion decreased biliary 3-glcA-LC excretion in EHBR (Fig. 3), biliary excretion of 3-glcA-LC in 60 min was 6.49 2.4% in the presence of DBSP infusion (P B0.05 vs. control). 3,7-Sul-UDC infusion had no effect on biliary 3-sul-C-tau excretion in EHBR (Fig. 4); biliary
Fig. 2. Effect of 3,7-sul-UDC infusion on bile flow and biliary [14C]3-glcA-LC excretion in EHBR. Data are means 9 S.D. of five control experiments (open circles) and three experiments of 3,7-sul-UDC infusion at the rate of 0.2 mmol/min per 100 g b. wt. started 30 min before 3-glcA-LC injection (closed circles).
excretion of 3-sul-C-tau in 60 min was 789 9.5 and 89 913% in the presence and absence of 3,7-sul-UDC infusion, respectively. 4. Discussion
Fig. 1. Effect of 3,7-sul-UDC infusion on bile flow and biliary [14C]3-glcA-LC excretion in SDR. Data are means 9 S.D. of five control experiments (open circles) and three experiments of 3,7-sul-UDC infusion at the rate of 0.2 mmol/min per 100 g b. wt. started 30 min before 3-glcA-LC injection (closed circles).
Although biliary excretion of both 3-glcA-LC and 3,7-sul-UDC was markedly impaired in EHBR [1,7], the infusion of 3,7-sul-UDC had no effect on biliary 3-glcA-LC excretion in SDR (Fig. 1). These data suggest that biliary excretory pathways of 3-glcA-LC and 3,7-sul-UDC are different, since the infusion rate of 3,7-sul-UDC in the present study (0.2 mmol/min per 100 g b.wt.) was above its excretory maximum, which is estimated to be approximately 0.1 mmol/min per 100 g b.wt. [24]. Although a defect of Mrp2 was reported in EHBR [3,4], the results of the present study as well as previous studies [20,21,25,26] suggest that the impaired biliary excretion of some organic anions and bile acid conjugates is due to the secondary effect of the Mrp2 defect.
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The enhanced biliary excretion of 3-glcA-LC by 3,7-sul-UDC observed in EHBR (Fig. 2) is considered to be due to 1) the displacement of 3-glcALC bound to albumin, thus increasing its uptake by the liver, 2) enhanced sinusoidal uptake of 3-glcA-LC, 3) the displacement of 3-glcA-LC bound to cytosolic binders, which are considered to be important for intrahepatic transport, and 4) the interaction of canalicular transporters for 3glcA-LC and 3,7-sul-UDC. We previously reported a similar enhancement of biliary 3-sul-LC excretion by 3,7-sul-UDC and an absence of an effect by 3,7-sul-UDC on serum clearance of 3sul-LC in EHBR [14]. Unfortunately, the amount of [14C]3-glcA-LC was not enough to study its serum clearance in the present study. However, since the canalicular transport is considered to be a rate limiting step of hepatobiliary transport of organic anions, an enhancement of the affinity for 3-glcA-LC of a canalicular transporter which is present in EHBR, an Mrp2-defective rat, due to a conformational change of the transporter is the
Fig. 3. Effect of DBSP infusion on bile flow and biliary [14C]3-glcA-LC excretion in EHBR. Data are means 9 S.D. of five control experiments (open circles) and three experiments of 3,7-sul-UDC infusion at the rate of 0.2 mmol/min per 100 g b. wt. started 30 min before 3-glcA-LC injection (closed circles).
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Fig. 4. Effect of 3,7-sul-UDC infusion on bile flow and biliary [14C]3-sul-C-tau excretion in EHBR. Data are means 9 S.D. of five control experiments (open circles) and three experiments of 3,7-sul-UDC infusion at the rate of 0.2 mmol/min per 100 g b. wt. started 30 min before 3-sul-C-tau injection (closed circles).
most likely mechanism of the enhanced 3-glcALC excretion by 3,7-sul-UDC. A report by Niinuma et al. that E3040-sulfate enhanced the transport of S-(2,4-dinitrophenyl)-glutathione by canalicular membrane vesicles of SDR [25] further supports such possibility of a conformational change of an unknown transporter. In contrast to the results with 3,7-sul-UDC, DBSP inhibited biliary 3-glcA-LC excretion in EHBR (Fig. 3). We previously reported that biliary excretion of DBSP was markedly reduced in EHBR and that DBSP inhibited biliary 3-sul-LC excretion both in SDR and EHBR [9]. Accordingly, DBSP is considered to compete to canalicular 3-glcA-LC transport by unknown transporter(s), which is present in EHBR. In contrast to the results with 3-glcA-LC (Fig. 2) and 3-sul-LC [14], biliary 3-sul-C-tau excretion was not affected by 3,7-sul-UDC in EHBR (Fig. 4). We previously reported that the extent of the impairment of biliary excretion of 3-sul-C-tau was not so prominent as that of 3-sul-LC-tau [17] and
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3-glcA-LC [1], suggesting that the involvement of Mrp2 on canalicular 3-sul-C-tau excretion is smaller than that of 3-sul-LC-tau and 3-glcA- LC. The absence of the effect of 3,7-sul-UDC on 3-sul-C-tau excretion in EHBR suggests a complete difference of the canalicular transport of these hydrophilic bile acid conjugates in the absence of Mrp2. In conclusion, 3,7-sul-UDC may interact to the excretory pathway of 3-glcA-LC, possibly by a confirmational change of an unknown canalicular transporter, and enhances biliary excretion of 3glcA-LC in EHBR.
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