Hepatic uptake and efflux processes involved in bile formation are maintained by distinct transport systems expressed at the two polar surface domains of liver cells. After canalicular secretion, bile composition undergoes further modification in the bile canaliculi, involving reabsorption and secretion processes maintained by apical and basolateral transport system in cholangiocytes. Figure 1 shows a scheme of hepatocellular and bile ductular transport proteins involved in uptake and efflux of endogenous and exogenous (xenobiotic) cholephilic compounds.
Hepatic Transport Systems
Properties and Function of Basolateral (Sinusoidal) Transporters.
Sodium-dependent and sodium-independent transport pathways have been identified to play a key role in hepatic uptake of endogenous and exogenous substances from sinusoidal blood plasma (Fig. 1). The sodium-dependent pathway is represented by the sodium taurocholate cotransporting polypeptide NTCP (SLC10A1) (reviewed by Hagenbuch and Dawson5), the substrate specificity of which is essentially limited to conjugated bile salts and certain sulfated steroids. NTCP accounts for more than 80% of conjugated (i.e., taurocholate and glycocholate) but less than 50% of unconjugated (i.e., cholate) bile salt uptake.5 In contrast, the sodium-independent pathway is represented by different members of the superfamily of organic anion-transporting polypeptides (OATP/SLCO) (reviewed by Hagenbuch and Meier6). In human liver, the highest expressions are found for OATP1B1 (SLCO1B1) and its 80% sequence homologue OATP1B3 (SLCO1B3), both of which are predominantly if not exclusively expressed in the liver. With the exception of OATP2B1 (SLCO2B1), the substrate specificity of which seems to be limited to bromosulphophtalein and steroid sulfates, OATP1A2 (SLCO1A2), OATP1B1, and OATP1B3 exhibit overlapping transport activities for conjugated and unconjugated bile salts, bromosulphophtalein, neutral steroids, steroid sulfates and glucuronides, and selected organic cations.6 Furthermore, numerous drugs are substrates of OATPs, including the antihistamine fexofenadine, opioid peptides, digoxin, the HMG CoA-reductase inhibitor pravastatin, the angiotensin-converting enzyme inhibitor enalapril, and the antimetabolite methotrexate.6 In addition, OATP1B1 and OATP1B3 mediate the uptake of the hepatotoxins phalloidin and microcystin into human liver,7–9 while hepatic uptake of amanitin, the most dangerous natural toxins causing hepatic failure seems to be exclusively mediated by OATP1B3.10
In addition, the sodium-independent uptake systems involve the organic anion and organic cation transporter family of solute carriers (SLC22), belonging to a gene family separate from the OATPs. OAT2 (SLC22A7) is the only transporter of the OAT/OCT family expressed in human liver and is believed to be liver-specific (reviewed by Koepsell and Endou11). OCT1 (SCL22A1) is expressed in human liver as well as in kidneys, small intestine and colon.11 In contrast to hepatic OATPs the exact role of OAT2 and OCT1 for hepatic uptake of drugs and bile constituents remains to be established.
In addition to these uptake systems, the basolateral hepatocyte membrane also localizes several adenosine triphosphate (ATP)-dependent efflux pumps. These transporters belong to the family of multidrug resistance–associated proteins (MRPs) (ABCC), which are multispecific transporters for different organic anions (reviewed by Homolya et al.12). Among the MRP family of ATP-binding cassette (ABC) transporters, MRP1 (ABCC1), MRP3 (ABCC3), MRP4 (ABCC4), MRP5 (ABCC5), and MRP6 (ABCC6) have been implicated in the cellular efflux of drug-glutathione, -glucuronide, and -sulfate conjugates (MRP1); the efflux of bile salts (MRP3); and the transport of nucleoside analog drugs such as zidovudine, lamivudine, and stavudine (MRP4) and cyclic adenosine and guanosine monophosphate, as well as methotrexate and the purine analogs 6-mercaptopurine and 6-thioguanine (MRP4 and MRP5).13 The physiological substrates of MRP6 are yet unknown.
Regulation of Basolateral Transporters.
Hepatocellular transport systems are subject to extensive transcriptional and posttranscriptional regulation, allowing adaptational changes in response to the intracellular accumulation of bile salts (reviewed by Eloranta and Kullak-Ublick14 and Trauner and Boyer15). During cholestasis, the sodium taurocholate–cotransporting polypeptide NTCP is suppressed through farnesoid X receptor (FXR)-mediated induction of small heterodimeric partner 1, thereby preventing the hepatocyte from further accumulating toxic bile salts.16, 17 Similarly, the expression of OATP1B1 is downregulated during cholestasis through bile acid–mediated activation of small heterodimeric partner 1, which leads to a repression of hepatocyte nuclear factor 1α, the major transcriptional activator of OATP1B1.18, 19 In contrast, cholestasis leads to an FXR-mediated activation of hepatic OATP1B3,20 which might constitute an escape mechanism promoting the hepatocellular clearance of xenobiotics during cholestasis. On the posttranscriptional level, sodium-dependent and -independent hepatocellular uptake systems are mainly regulated by cyclic adenosine monophosphate–mediated dephosphorylation processes, which is controlled by phophoinositide-3-kinase/protein kinase B.21–23 Furthermore, PDZK1 was demonstrated to be a critical determinant for the proper subcellular localization and function of rat Oatp1a1.24
The transcriptional regulation of basolaterally expressed MRPs is not fully elucidated. Studies in mice support the notion that Mrp3 and Mrp4 are induced through a pregnane X receptor–mediated pathway.25
Properties and Function of Apical (Canalicular) Transporters.
The secretion of bile salts and xenobiotics across the canalicular membrane of hepatocytes is mediated by various ABC transporters (Fig. 1). With the exception of FIC1 (ATP8B1), which is thought to play a role in the regulation of the enterohepatic bile acid pool and in the elimination of hydrophobic substances from the enterohepatic circulation,26 canalicular transporters involved in bile formation and hepatic drug clearance belong to different members of the superfamily of ABC transporters. These include members of the family of multidrug resistance (MDR) P-glycoproteins such as MDR1 (ABCB1), MDR3 (ABCB4), and the bile salt export pump (BSEP) (ABCB11). In addition, the canalicular membrane localizes the multidrug resistance–associated protein 2 (MRP2) (ABCC2) and the ABC half transporters breast cancer resistance protein (BCRP) (ABCG2) and the cholesterol flippase ABCG5 and ABCG8 (ABCG5 and ABCG8).
Within the family of multidrug resistance proteins, BSEP and MDR3 are two highly conserved members, which are involved in the secretion of cholephilic compounds from the liver cell into the bile canaliculus (reviewed by Kullak-Ublick27 and Meier and Stieger28). BSEP constitutes the predominant bile salt efflux system of hepatocytes and mediates the cellular excretion of numerous conjugated bile salts such as taurine- or glycine-conjugated cholate, chenodeoxycholate, and deoxycholate.29, 30 MDR3 was shown to function as an ATP-dependent phospholipid flippase, translocating phosphatidylcholine from the inner to the outer leaflet of the canalicular membrane (reviewed by van Erpecum31). Canalicular phospholipids are then solubilized by canalicular bile salts to form mixed micelles, thereby protecting cholangiocytes from the detergent properties of bile salts. In addition to these processes, MRP2, the only canalicular member of the multidrug resistance–associated protein family, mediates the canalicular transport of glucuronidated and sulfated bile salts. MRP2 is the main driving force for bile salt–independent bile flow through canalicular excretion of reduced glutathione. Furthermore, MRP2 transports a wide spectrum of organic anions, including bilirubin diglucuronide, glutathione conjugates, leukotriene C4, and divalent bile salt conjugates, as well as drug substrates such as cancer chemotherapeutic agents, uricosurics, and antibiotics (reviewed by Borst et al.32).
The exact contribution of MDR1 to hepatic bile formation remains to be established, but it is thought to contribute to the canalicular excretion of drugs and other xenobiotics into bile. Its broad substrate specificity and its physiological expression in various tissues with excretory and protective functions make MDR1 one of the major determinants of drug disposition and toxicity. Substrates are neutral and positively charged organic compounds and include various chemotherapeutic and immunosuppressant agents, antiarrhythmic drugs, HIV protease inhibitors, and antifungals.33, 34
The ABC half transporter breast cancer resistance protein BCRP (ABCG2) shows the highest expression levels in mammary epithelium and placenta, where it plays an important role in conferring a multidrug resistance phenotype against a variety of xenobiotics. Recently, BCRP has been shown to transport sulfated bile salt conjugates such as taurolithocholate sulfate in vitro35 and to be involved in the biliary excretion of drugs such as pitavastatin.36 It might therefore be speculated that BCRP contributes to the hepatocellular excretion of bile salts and xenobiotics. Furthermore, the heterodimeric transporter ABCG5/ABCG8 (ABCG5 and ABCG8) has been identified as the apical transport system involved in the hepatobiliary excretion of plant sterols and cholesterol (reviewed by Klett and Patel37 and Kosters et al.38). Overexpression of ABCG5/ABCG8 in transgenic mice led to an increase in biliary cholesterol secretion and a reduced intestinal absorption of dietary cholesterol, providing strong evidence for ABCG5/ABCG8 being involved in hepatocellular secretion and intestinal efflux of cholesterol.37 However, the possible role of these ABC half transporters for hepatic drug clearance and the development of drug-induced cholestasis remain to be determined.
Regulation of Canalicular Transporters.
Transcriptional regulation of BSEP and MDR3 is mediated by FXR.39–41 FXR-mediated activation of BSEP and MDR3 leads to increased bile salt efflux and the formation of mixed micelles in the biliary tree during cholestatic episodes, thereby preventing toxic effects of bile salts on hepatocytes and cholangiocytes. In addition, FXR has been shown to induce MRP2 expression in human hepatocytes, which might constitute another compensatory mechanism during cholestasis.42 In contrast, MDR1 is upregulated via the pregnane X receptor, which in addition to endogenous ligands is activated by different xenobiotics such as rifampicin or St. John's Wort.43–45 This pathway is part of a general cellular detoxification mechanism, because MDR1 is the key transporter protein involved in the cellular efflux of numerous drugs and xenobiotics. Transcriptional regulation of BCRP most likely involves the aryl hydrocarbon receptor and the epidermal growth factor, whereas ABCG5 and ABCG8 are direct targets of the oxysterol-dependent liver X receptor α and β.46
Posttranscriptional targeting of Bsep, Mdr2 (the rat homologue of human MDR3), and Mrp2 to the canalicular membrane47–49 is mediated by phophoinositide-3-kinase and protein kinase C isoforms.
Bile Ductular Transporters
Properties and Function of Basolateral and Apical Cholangiocyte Transporters.
Uptake of bile salts from canalicular bile into cholangiocytes is mediated by the apical sodium-dependent bile salt transporter (SCL10A2) (Fig. 1). The apical sodium-dependent bile salt transporter belongs to the superfamily of solute carriers and is identical with the gene product expressed in the terminal ileum of the small intestine.5 Furthermore, the uptake of bile salts involves the organic anion-transporting polypeptide 1A2 (OATP1A2), which belongs to the OATP superfamily of sodium-independent solute transporters (SCLO; former nomenclature SLC21).6
After their uptake into cholangiocytes, bile salts are effluxed at the basolateral cholangiocyte membrane into the peribiliary plexus via an anion exchange mechanism.50 From here, bile salts reach the portal circulation and undergo the cholehepatic shunt pathway. MRP3, a basolaterally expressed member of the family of multidrug resistance–associated proteins contributes to the efflux of bile salts from cholangiocytes.51 Moreover, MRP2 was recently localized in gallbladder-derived biliary epithelial cells, where it might contribute to taurocholate homeostasis.51 In addition, a splicing variant of rat apical sodium-dependent bile salt transporter could be localized to the basolateral membrane of cholangiocytes, where it is proposed to function as a bile salt efflux protein. However, the contribution of this truncated protein to bile salt efflux in human cholangiocytes has not been established.52 Furthermore, the heterodimeric organic solute transporter OSTα/OSTβ was recently found to be expressed in the basolateral membrane of cholangiocytes, where it is thought to contribute to bile acid and sterol transport into the peribiliary plexus.53