Canalicular bile formation: beyond single transporter functions
2002; Elsevier BV; Volume: 37; Issue: 2 Linguagem: Inglês
10.1016/s0168-8278(02)00203-9
ISSN1600-0641
Autores Tópico(s)Pharmacological Effects and Toxicity Studies
ResumoHepatic bile formation is governed by distinct transport systems expressed at the sinusoidal (basolateral) and canalicular surface domains of hepatocytes [[1]Meier P.J. Stieger B. Bile salt transporters.Annu Rev Physiol. 2002; 64: 635-661Crossref PubMed Scopus (465) Google Scholar]. Bile salts are predominantly taken up into hepatocytes by the Na+-dependent taurocholate cotransport protein (rodents: Ntcp, humans: NTCP; gene symbols Slc10a1/SLC10A1) and secreted into bile canaliculi by the bile salt export pump (rodents: Bsep (Abcb11); humans: BSEP (ABCB11)), a member of the ATP-binding-cassette (ABC) transporter superfamily [[1]Meier P.J. Stieger B. Bile salt transporters.Annu Rev Physiol. 2002; 64: 635-661Crossref PubMed Scopus (465) Google Scholar]. Biliary excretion of other amphipathic and albumin-bound organic anions is mediated by sinusoidal Na+-independent organic anion transporting polypeptides (rodents: Oatp1 (Slc21a1), Oatp2 (Slc21a5), Oatp4 (Slc21a10); humans: OATP-B (SLC21A9), OATP-C (SLC21A), OATP8 (SLC21A8) and the canalicular multidrug resistance-associated protein 2 (rodents: Mrp2 (Abcc2); humans: MRP2 (ABCC2)) [[2]Kullak-Ublick G.A. Stieger B. Hagenbuch B. Meier P.J. Hepatic transport of bile salts.Semin Liver Dis. 2000; 20: 273-292Crossref PubMed Scopus (234) Google Scholar]. Mrp2/MRP2 mediates ATP-dependent canalicular excretion of a wide range of conjugated organic anions including bilirubin diglucuronide, sulfated bile salts and glutathione (GSH) S-conjugates [[3]Keppler D. König J. Hepatic secretion of conjugated drugs and endogenous substances.Semin Liver Dis. 2000; 20: 265-272Crossref PubMed Scopus (211) Google Scholar]. Furthermore, Mrp2 induces canalicular GSH secretion and, thus, represents an important mediator of bile-salt independent bile flow [[4]Oude Elferink R.P. Ottenhof R. Liefting W. de Haan J. Jansen P.L. Hepatobiliary transport of glutathione and glutathione conjugate in rats with hereditary hyperbilirubinemia.J Clin Invest. 1989; 84: 476-483Crossref PubMed Google Scholar]. Defective expression of canalicular Mrp2/MRP2 results in conjugated hyperbilirubinemia in transport mutant (TR−) rats [[5]Paulusma C.C. Bosma P.J. Zaman G.J. Bakker C.T. Otter M. Scheffer G.L. et al.Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene.Science. 1996; 271: 1126-1128Crossref PubMed Scopus (795) Google Scholar] and forms the basis of the Dubin–Johnson syndrome in humans [[3]Keppler D. König J. Hepatic secretion of conjugated drugs and endogenous substances.Semin Liver Dis. 2000; 20: 265-272Crossref PubMed Scopus (211) Google Scholar]. Finally, the canalicular membrane localizes the ATP-dependent multidrug-resistance protein 2/3 (rodents: Mdr2 (Abcb4); humans: MDR3 (ABCB4)), which mediates translocation of phosphatidylcholine (PC) from the inner to the outer leaflet of the canalicular membrane [[6]Oude Elferink R.P.J. Groen A.K. Mechanisms of biliary lipid secretion and their role in lipid homeostasis.Semin Liver Dis. 2000; 20: 293-305Crossref PubMed Google Scholar]. Intracanalicular bile salts then solubilize PC and cholesterol from the outer canalicular membrane leaflet in the form of vesicles and mixed micelles [[6]Oude Elferink R.P.J. Groen A.K. Mechanisms of biliary lipid secretion and their role in lipid homeostasis.Semin Liver Dis. 2000; 20: 293-305Crossref PubMed Google Scholar]. These mixed lipid–bile salt aggregates protect the biliary epithelium from the detergent properties of bile salts. Lack of Mdr2 results in progressive bile ductular damage and cholestasis in knockout mice [[7]Smit J.J.M. Schinkel A.H. Oude Elferink R.P.J. Groen A.K. Wagenaar E. van Deemter L. et al.Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease.Cell. 1993; 75: 451-463Abstract Full Text PDF PubMed Scopus (1318) Google Scholar]. In humans, defective expression of BSEP or MDR3 is associated with progressive familial intrahepatic cholestasis type2 (PFIC2) or type 3 (PFIC3), respectively. Functional impairments of BSEP and/or MDR3 have also been proposed as causative mechanisms for certain acquired forms of cholestasis including drug-induced cholestasis [[8]Stieger B. Fattinger K. Madon J. Kullak-Ublick G.A. Meier P.J. Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver.Gastroenterology. 2000; 118: 422-430Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar] and intrahepatic cholestasis of pregnancy [[9]Jacquemin E. de Vree J.M.L. Cresteil D. Sokal E.M. Sturm E. Dumont M. et al.The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood.Gastroenterology. 2001; 120: 1448-1458Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar].Drugs that have been suggested to cause cholestasis through direct cis-inhibition of Bsep function include cyclosporine, glibenclamide, rifampicin, bosentan and troglitazone [8Stieger B. Fattinger K. Madon J. Kullak-Ublick G.A. Meier P.J. Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver.Gastroenterology. 2000; 118: 422-430Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 10Fattinger K. Funk C. Pantze M. Weber C. Reichen J. Stieger B. Meier P.J. The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions.Clin Pharmacol Ther. 2001; 69: 223-231Crossref PubMed Scopus (427) Google Scholar, 11Funk C. Ponelle C. Scheuermann G. Pantze M. Cholestatic potential of troglitazone as a possible factor contributing to troglitazone-induced hepatotoxicity: In vivo and in vitro interaction at the canalicular bile salt export pump (Bsep) in the rat.Mol Pharmacol. 2001; 59: 627-635Crossref PubMed Scopus (275) Google Scholar]. In contrast, the cholestatic estrogen-metabolite estradiol-17β-glucuronide must first be excreted into bile canaliculi by Mrp2 before it can transinhibit Bsep function from the canalicular side [[8]Stieger B. Fattinger K. Madon J. Kullak-Ublick G.A. Meier P.J. Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver.Gastroenterology. 2000; 118: 422-430Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar]. Alternatively, estradiol-17β-glucuronide induced inhibition of Bsep could result also from a direct intramembranous interaction between Mrp2 and Bsep [[12]Huang L. Smit J.W. Meijer D.K. Vore M. Mrp2 is essential for estradiol-17beta (beta-D-glucuronide)-induced cholestasis in rats.Hepatology. 2000; 32: 66-72Crossref PubMed Scopus (102) Google Scholar]. Although the exact mechanism remains to be elucidated, it appears clear that effective inhibition of Bsep by estradiol-17β-glucuronide requires intact canalicular Mrp2.In this issue of the Journal, Fouassier et al. provide a further and surprising example of Mrp2-dependent disruption of coordinated canalicular bile salt, phospholipid and cholesterol secretion [[13]Fouassier L. Kinnman N. Lefèvre G. Lasnier E. Rey C. Poupon R. et al.Contribution of mrp2 in alterations of canalicular bile formation by the endothelin antagonist bosentan.J Hepatol. 2002; 37: 184-191Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]. They treated anesthetized rats with biliary fistulas with increasing concentrations of the endothelin EtA/B receptor antagonist bosentan and analyzed its effects on bile flow and biliary excretion of glutathione, bicarbonate, bilirubin, bile salts, phospholipids and cholesterol. Infusions of bosentan, at concentrations without any effects on the hepatic microcirculation, induced a marked choleresis that was associated with increased biliary excretion of glutathione, bicarbonate and bilirubin. In contrast, while taurocholate secretion was only slightly decreased, bosentan treatment markedly reduced the biliary output of phospholipids and cholesterol. Surprisingly, neither choleresis nor the strong uncoupling effect of bosentan on canalicular bile salt and phospholipid secretion was observed in Mrp2 deficient TR− rats, whereas reduced biliary cholesterol excretion was still maintained to some extent. These studies clearly indicate that bosentan induces bile-salt independent bile flow through functional activation of Mrp2, which is in line with previous findings of endothelin1 and endothelin receptor antagonists in renal proximal tubules of the killifish [[14]Masereeuw R. Terlouw S.A. Van Aubel R.A.M.H. Russel F.G.M. Miller D.S. Endothelin B receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule.Mol Pharmacol. 2000; 57: 59-67PubMed Google Scholar]. As a consequence of Mrp2 induced choleresis and bilirubin output bile salts might be displaced and diluted within bile canaliculi below the critical concentration for solubilization of canalicular phospholipids and cholesterol. Furthermore, the persistent uncoupling effect on cholesterol even in the absence of Mrp2 might indicate an additional, as yet unknown, effect of bosentan on biliary cholesterol excretion.These carefully conducted studies, together with previous demonstrations of stimulation of Mrp2 by low concentrations of drugs such as sulfinpyrazone, indomethacin and penicillin [15Bakos E. Evers R. Sinko E. Varadi A. Borst P. Sarkadi B. Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions.Mol Pharmacol. 2000; 57: 760-768Crossref PubMed Scopus (288) Google Scholar, 16Evers R. de Haas M. Sparidans R. Beijnen J. Wielinga P.R. Lankelma J. et al.Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export.Br J Cancer. 2000; 83: 375-383Crossref PubMed Scopus (208) Google Scholar], underline that drug-induced alterations of bile formation and bile composition can be accounted for by alterations of unexpected carrier functions. Hence, decreased biliary phospholipid secretion does not necessary mean a defect in the canalicular phospholipid translocase Mdr2 or MDR3, but can be also a consequence of an intracanalicular physicochemical disequilibrium caused by increased Mrp2-dependent organic anion secretion. Whether the same is also true for cholesterol remains to be investigated in more detail. Nevertheless, since reduced phospholipid secretion increases the detergent properties of bile salts within the biliary tree, stimulation of Mrp2-mediated transport functions could well represent an important cause of acquired forms of cholestatic liver disease such as, for example, drug-induced cholangiopathies. Furthermore, the studies of Fouassier and coworkers remind us that careful in vivo assessment of carrier functions is at least as, or even more, important as mere screening for carrier protein expression under various physiologic and pathophysiologic situations. And finally, we are reminded that functional alterations of a selective, but primarily unexpected, carrier can induce physicochemical changes in the primary bile fluid that might well be of greater, albeit somewhat hidden, pathophysiologic significance than is commonly assumed. Thus, Fouassier and coworkers have to be congratulated for the careful conductance of their study and for extending their investigations to the Mrp2-deficient TR− rats. It will now be interesting to see to what extent the observed hepatobiliary effects of bosentan relate to its intrinsic endothelin receptor antagonist activity, or whether bosentan and/or its metabolites are just an addition to the increasing list of drugs that are Mrp2 substrates and can stimulate Mrp2 at low concentrations. Hepatic bile formation is governed by distinct transport systems expressed at the sinusoidal (basolateral) and canalicular surface domains of hepatocytes [[1]Meier P.J. Stieger B. Bile salt transporters.Annu Rev Physiol. 2002; 64: 635-661Crossref PubMed Scopus (465) Google Scholar]. Bile salts are predominantly taken up into hepatocytes by the Na+-dependent taurocholate cotransport protein (rodents: Ntcp, humans: NTCP; gene symbols Slc10a1/SLC10A1) and secreted into bile canaliculi by the bile salt export pump (rodents: Bsep (Abcb11); humans: BSEP (ABCB11)), a member of the ATP-binding-cassette (ABC) transporter superfamily [[1]Meier P.J. Stieger B. Bile salt transporters.Annu Rev Physiol. 2002; 64: 635-661Crossref PubMed Scopus (465) Google Scholar]. Biliary excretion of other amphipathic and albumin-bound organic anions is mediated by sinusoidal Na+-independent organic anion transporting polypeptides (rodents: Oatp1 (Slc21a1), Oatp2 (Slc21a5), Oatp4 (Slc21a10); humans: OATP-B (SLC21A9), OATP-C (SLC21A), OATP8 (SLC21A8) and the canalicular multidrug resistance-associated protein 2 (rodents: Mrp2 (Abcc2); humans: MRP2 (ABCC2)) [[2]Kullak-Ublick G.A. Stieger B. Hagenbuch B. Meier P.J. Hepatic transport of bile salts.Semin Liver Dis. 2000; 20: 273-292Crossref PubMed Scopus (234) Google Scholar]. Mrp2/MRP2 mediates ATP-dependent canalicular excretion of a wide range of conjugated organic anions including bilirubin diglucuronide, sulfated bile salts and glutathione (GSH) S-conjugates [[3]Keppler D. König J. Hepatic secretion of conjugated drugs and endogenous substances.Semin Liver Dis. 2000; 20: 265-272Crossref PubMed Scopus (211) Google Scholar]. Furthermore, Mrp2 induces canalicular GSH secretion and, thus, represents an important mediator of bile-salt independent bile flow [[4]Oude Elferink R.P. Ottenhof R. Liefting W. de Haan J. Jansen P.L. Hepatobiliary transport of glutathione and glutathione conjugate in rats with hereditary hyperbilirubinemia.J Clin Invest. 1989; 84: 476-483Crossref PubMed Google Scholar]. Defective expression of canalicular Mrp2/MRP2 results in conjugated hyperbilirubinemia in transport mutant (TR−) rats [[5]Paulusma C.C. Bosma P.J. Zaman G.J. Bakker C.T. Otter M. Scheffer G.L. et al.Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene.Science. 1996; 271: 1126-1128Crossref PubMed Scopus (795) Google Scholar] and forms the basis of the Dubin–Johnson syndrome in humans [[3]Keppler D. König J. Hepatic secretion of conjugated drugs and endogenous substances.Semin Liver Dis. 2000; 20: 265-272Crossref PubMed Scopus (211) Google Scholar]. Finally, the canalicular membrane localizes the ATP-dependent multidrug-resistance protein 2/3 (rodents: Mdr2 (Abcb4); humans: MDR3 (ABCB4)), which mediates translocation of phosphatidylcholine (PC) from the inner to the outer leaflet of the canalicular membrane [[6]Oude Elferink R.P.J. Groen A.K. Mechanisms of biliary lipid secretion and their role in lipid homeostasis.Semin Liver Dis. 2000; 20: 293-305Crossref PubMed Google Scholar]. Intracanalicular bile salts then solubilize PC and cholesterol from the outer canalicular membrane leaflet in the form of vesicles and mixed micelles [[6]Oude Elferink R.P.J. Groen A.K. Mechanisms of biliary lipid secretion and their role in lipid homeostasis.Semin Liver Dis. 2000; 20: 293-305Crossref PubMed Google Scholar]. These mixed lipid–bile salt aggregates protect the biliary epithelium from the detergent properties of bile salts. Lack of Mdr2 results in progressive bile ductular damage and cholestasis in knockout mice [[7]Smit J.J.M. Schinkel A.H. Oude Elferink R.P.J. Groen A.K. Wagenaar E. van Deemter L. et al.Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease.Cell. 1993; 75: 451-463Abstract Full Text PDF PubMed Scopus (1318) Google Scholar]. In humans, defective expression of BSEP or MDR3 is associated with progressive familial intrahepatic cholestasis type2 (PFIC2) or type 3 (PFIC3), respectively. Functional impairments of BSEP and/or MDR3 have also been proposed as causative mechanisms for certain acquired forms of cholestasis including drug-induced cholestasis [[8]Stieger B. Fattinger K. Madon J. Kullak-Ublick G.A. Meier P.J. Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver.Gastroenterology. 2000; 118: 422-430Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar] and intrahepatic cholestasis of pregnancy [[9]Jacquemin E. de Vree J.M.L. Cresteil D. Sokal E.M. Sturm E. Dumont M. et al.The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood.Gastroenterology. 2001; 120: 1448-1458Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar]. Drugs that have been suggested to cause cholestasis through direct cis-inhibition of Bsep function include cyclosporine, glibenclamide, rifampicin, bosentan and troglitazone [8Stieger B. Fattinger K. Madon J. Kullak-Ublick G.A. Meier P.J. Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver.Gastroenterology. 2000; 118: 422-430Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 10Fattinger K. Funk C. Pantze M. Weber C. Reichen J. Stieger B. Meier P.J. The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions.Clin Pharmacol Ther. 2001; 69: 223-231Crossref PubMed Scopus (427) Google Scholar, 11Funk C. Ponelle C. Scheuermann G. Pantze M. Cholestatic potential of troglitazone as a possible factor contributing to troglitazone-induced hepatotoxicity: In vivo and in vitro interaction at the canalicular bile salt export pump (Bsep) in the rat.Mol Pharmacol. 2001; 59: 627-635Crossref PubMed Scopus (275) Google Scholar]. In contrast, the cholestatic estrogen-metabolite estradiol-17β-glucuronide must first be excreted into bile canaliculi by Mrp2 before it can transinhibit Bsep function from the canalicular side [[8]Stieger B. Fattinger K. Madon J. Kullak-Ublick G.A. Meier P.J. Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver.Gastroenterology. 2000; 118: 422-430Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar]. Alternatively, estradiol-17β-glucuronide induced inhibition of Bsep could result also from a direct intramembranous interaction between Mrp2 and Bsep [[12]Huang L. Smit J.W. Meijer D.K. Vore M. Mrp2 is essential for estradiol-17beta (beta-D-glucuronide)-induced cholestasis in rats.Hepatology. 2000; 32: 66-72Crossref PubMed Scopus (102) Google Scholar]. Although the exact mechanism remains to be elucidated, it appears clear that effective inhibition of Bsep by estradiol-17β-glucuronide requires intact canalicular Mrp2. In this issue of the Journal, Fouassier et al. provide a further and surprising example of Mrp2-dependent disruption of coordinated canalicular bile salt, phospholipid and cholesterol secretion [[13]Fouassier L. Kinnman N. Lefèvre G. Lasnier E. Rey C. Poupon R. et al.Contribution of mrp2 in alterations of canalicular bile formation by the endothelin antagonist bosentan.J Hepatol. 2002; 37: 184-191Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]. They treated anesthetized rats with biliary fistulas with increasing concentrations of the endothelin EtA/B receptor antagonist bosentan and analyzed its effects on bile flow and biliary excretion of glutathione, bicarbonate, bilirubin, bile salts, phospholipids and cholesterol. Infusions of bosentan, at concentrations without any effects on the hepatic microcirculation, induced a marked choleresis that was associated with increased biliary excretion of glutathione, bicarbonate and bilirubin. In contrast, while taurocholate secretion was only slightly decreased, bosentan treatment markedly reduced the biliary output of phospholipids and cholesterol. Surprisingly, neither choleresis nor the strong uncoupling effect of bosentan on canalicular bile salt and phospholipid secretion was observed in Mrp2 deficient TR− rats, whereas reduced biliary cholesterol excretion was still maintained to some extent. These studies clearly indicate that bosentan induces bile-salt independent bile flow through functional activation of Mrp2, which is in line with previous findings of endothelin1 and endothelin receptor antagonists in renal proximal tubules of the killifish [[14]Masereeuw R. Terlouw S.A. Van Aubel R.A.M.H. Russel F.G.M. Miller D.S. Endothelin B receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule.Mol Pharmacol. 2000; 57: 59-67PubMed Google Scholar]. As a consequence of Mrp2 induced choleresis and bilirubin output bile salts might be displaced and diluted within bile canaliculi below the critical concentration for solubilization of canalicular phospholipids and cholesterol. Furthermore, the persistent uncoupling effect on cholesterol even in the absence of Mrp2 might indicate an additional, as yet unknown, effect of bosentan on biliary cholesterol excretion. These carefully conducted studies, together with previous demonstrations of stimulation of Mrp2 by low concentrations of drugs such as sulfinpyrazone, indomethacin and penicillin [15Bakos E. Evers R. Sinko E. Varadi A. Borst P. Sarkadi B. Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions.Mol Pharmacol. 2000; 57: 760-768Crossref PubMed Scopus (288) Google Scholar, 16Evers R. de Haas M. Sparidans R. Beijnen J. Wielinga P.R. Lankelma J. et al.Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export.Br J Cancer. 2000; 83: 375-383Crossref PubMed Scopus (208) Google Scholar], underline that drug-induced alterations of bile formation and bile composition can be accounted for by alterations of unexpected carrier functions. Hence, decreased biliary phospholipid secretion does not necessary mean a defect in the canalicular phospholipid translocase Mdr2 or MDR3, but can be also a consequence of an intracanalicular physicochemical disequilibrium caused by increased Mrp2-dependent organic anion secretion. Whether the same is also true for cholesterol remains to be investigated in more detail. Nevertheless, since reduced phospholipid secretion increases the detergent properties of bile salts within the biliary tree, stimulation of Mrp2-mediated transport functions could well represent an important cause of acquired forms of cholestatic liver disease such as, for example, drug-induced cholangiopathies. Furthermore, the studies of Fouassier and coworkers remind us that careful in vivo assessment of carrier functions is at least as, or even more, important as mere screening for carrier protein expression under various physiologic and pathophysiologic situations. And finally, we are reminded that functional alterations of a selective, but primarily unexpected, carrier can induce physicochemical changes in the primary bile fluid that might well be of greater, albeit somewhat hidden, pathophysiologic significance than is commonly assumed. Thus, Fouassier and coworkers have to be congratulated for the careful conductance of their study and for extending their investigations to the Mrp2-deficient TR− rats. It will now be interesting to see to what extent the observed hepatobiliary effects of bosentan relate to its intrinsic endothelin receptor antagonist activity, or whether bosentan and/or its metabolites are just an addition to the increasing list of drugs that are Mrp2 substrates and can stimulate Mrp2 at low concentrations.
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