42.2.1.1

Influence of Albumin Binding

Studies performed in the isolated perfused rat liver revealed that as much as 50% of bilirubin or sulfobromophthalein (BSP) is removed from perfusate in a single pass. Despite the fact that these compounds are bound avidly to albumin, it is the free organic anion rather than the albumin-organic anion complex that is taken up. Subsequent investigations performed with overnight cultured rat hepatocytes revealed saturable BSP uptake that was inhibited by bilirubin. Similar to findings in the isolated perfused rat liver, ligand but not albumin was taken up by cultured hepatocytes; that is, albumin remained extracellular. The lack of specificity for albumin as a carrier was noted in studies in the isolated perfused rat liver in which there was no enhancement of bilirubin uptake when it was presented as a complex with albumin as compared to its presentation as a complex with its normally cytosolic binding protein ligandin or in the absence of albumin.

42.2.1.2

Driving Forces

The driving forces for nonbile acid organic anion uptake have also been the subject of a great deal of study. Early experiments performed in isolated rat hepatocytes indicated saturable uptake of BSP without a requirement for cellular energy, as determined by the lack of effect of preincubation of cells in antimycin A, rotenone, or carbonylcyanide m -chlorophenylhydrazone. However, subsequent studies using different metabolic inhibitors (DNP, KCN, or sodium azide/2-deoxyglucose) clearly showed that cellular energy was required for BSP uptake, and a similar difference in sensitivity of the uptake process to various metabolic inhibitors was also seen in studies of GSH-BSP uptake by isolated rat hepatocytes. The physiologic significance of this differential sensitivity remains unknown.

The development of a short-term cultured rat hepatocyte system facilitated studies in which the environment could be manipulated, and transport characteristics and driving forces could be examined. In particular, isosmotic substitution of NaCl in the medium by sucrose resulted in an 80% fall in uptake of BSP or bilirubin by cultured rat hepatocytes. Although this result might have signified a requirement for extracellular Na ⁺ , this was not the case. There was no significant effect on uptake by substitution of Na ⁺ in medium by K ⁺ , Li ⁺ , or choline. In contrast, when Cl ⁻ was replaced by HCO ₃ ⁻ or gluconate, ligand uptake was markedly reduced. Similar Cl ⁻ dependence has been described for hepatocyte uptake of bilirubin by cultured rat hepatocytes and by isolated perfused rat liver. The mechanism for this effect remains unclear. There was no stimulation of BSP uptake by unidirectional Cl ⁻ gradients, although the affinity of BSP for the surface of hepatocytes was approximately 10-fold higher in the presence of Cl ⁻ than in its absence. It is interesting to note that Cl ⁻ -dependent BSP uptake is seen in differentiated hepatocytes and is lost with time in culture and in hepatoma cell lines. As this Cl ⁻ -dependent extraction of BSP from albumin appeared to be characteristic of the high-affinity hepatocyte organic anion uptake mechanism, it was subsequently utilized to develop an assay to clone the transporter using a functional expression strategy in Xenopus laevis oocytes (see below). The influence of unidirectional pH gradients on BSP transport by cultured rat hepatocytes was also examined. These studies showed that BSP transport was stimulated by an inside-to-outside OH ⁻ gradient, consistent with OH ⁻ exchange or H ⁺ cotransport.

42.2.1.3

Physiologic Modulation of Transport

As noted above, it is possible for lipophilic molecules to cross a lipid bilayer without requiring protein facilitation. However, the specificity of the hepatocyte for uptake of many of these compounds and their reduced uptake during physiologic perturbations, as noted below, suggest that simple passage through lipid bilayers represents at best a minor part of their uptake. One example is the process of liver regeneration following two-thirds partial hepatectomy in the rat. Within 6 hours of the regenerative stimulus, influx of bilirubin or BSP is reduced by 50%, when calculated independently of liver mass, returning to normal levels by 4 days. Similar studies were performed with the hypolipidemic agent nafenopin, which when administered to rats for 2 days, results in liver hypertrophy and hyperplasia that morphologically resembles regeneration. Of note is the fact that influx of BSP and bilirubin glucuronides was reduced by 50% in these rats, while bilirubin influx was normal. This dissociation of uptake for these subclasses of organic anions suggests that they may have partially independent uptake mechanisms. Extracellular ATP also downregulates hepatocyte uptake of BSP, and this has been attributed to signal transduction mediated by a purinergic receptor that recognizes ATP ^{− 4.}

42.2.2.1

Biochemical Studies

The studies described above are consistent with the existence of an organic anion transporter(s) that is present on the basolateral (sinusoidal) plasma membrane of hepatocytes. The nature of this transporter has been the subject of investigation for several years. Initial studies described high-affinity binding of BSP to preparations of liver cell plasma membrane and a 55 kDa protein termed BSP/bilirubin binding protein was identified. Little has been done subsequently to characterize the potential physiologic role of this protein, and it must be borne in mind that functional studies were performed at high concentrations of ligand in the absence of albumin, suggesting that low-affinity transport was examined. In addition, this protein was shown to be present on the surface of HepG2 cells, a cell line that lacks the high-affinity transporter and is unable to take up BSP in the presence of albumin. Other studies isolated a 170 kDa protein termed bilitranslocase on the basis of its ability to transport BSP and bilirubin when incorporated into liposomes. However, this protein is not hepatocyte specific and is also present in HepG2 and vascular endothelial cells. Bilitranslocase was cloned from a rat liver expression library utilizing a monoclonal antibody. The derived protein sequence is unique and has been used in a number of functional studies. However, analysis of the nucleotide sequence (Genbank accession number Y12178) reveals that it is 94% identical to the sequence of the inverse strand of ceruloplasmin, suggesting that a cloning artifact likely occurred. A third group of studies identified a 55 kDa organic anion-binding protein (oabp) following photoaffinity labeling of rat liver plasma membrane preparations by ³⁵ S-BSP. However, this protein was shown to be identical to the ß-subunit of F ₁ -ATPase, an inner mitochondrial membrane protein. Although the protein appeared to have a form that could be identified on the plasma membrane of hepatocytes, its potential function in organic anion transport remains unknown.

42.2.2.2

Molecular Biology Studies

As discussed above, Cl ⁻ -dependent extraction of BSP from albumin is characteristic of the high-affinity hepatocyte organic anion uptake mechanism. This unique transport characteristic was used to clone the transporter using a functional expression strategy in X. laevis oocytes. A single cRNA was isolated which when injected into oocytes resulted in a substantial signal of BSP uptake activity that was suppressed by Cl ⁻ substitution. The corresponding cDNA encodes a rat liver protein that was named organic anion transporting polypeptide (oatp, now known as oatp1a1), the first member of the solute carrier organic anion transporter gene family Slco. Since the initial description of oatp1a1, over 20 additional members of the oatp family have been described. These proteins have a relatively high degree of amino acid similarity as well as overlap of transported substrates, although their tissue distributions are varied. In subsequent studies, we used oatp1a1 as a well-characterized, prototypical member of the oatp family. It mediates high-affinity transport of a diverse group of compounds including organic anions, such as sulfobromophthalein (BSP) and taurocholate, peptide drugs including the thrombin inhibitor CRC220 and the angiotensin-converting enzyme inhibitor enalapril, and various steroid hormones such as estradiol 17β-glucuronide. There is no clear common structural similarity of compounds that are substrates for any of the oatps, although pharmacophore modeling has provided some insights into substrate requirements. Transport by oatps is Na ⁺ -independent, involving exchange with an intracellular anion such as bicarbonate or glutathione. Computer analysis of oatp1a1 cDNA reveals an open reading frame of 2010 nucleotides, encoding a protein with a high degree of hydrophobicity. Although computer modeling predicts 12- as well as 10-transmembrane domain structures, glycosylation mapping and antigenic epitope accessibility in protein expressed in transfected cells and rat hepatocytes indicate that the 12-transmembrane domain model is correct and that the second and fifth extracellular loops are glycosylated at asparagines 124, 136, and 492. Oatp1a1 expression appears to be limited to hepatocytes and epithelial cells of the choroid plexus and the S3 segment of the proximal tubule.

As illustrated in Table 42.1 , several members of the oatp family are highly expressed in hepatocytes from rats, mice, and humans. These hepatocyte oatps are distributed on the basolateral plasma membrane and have overlapping substrate specificities. They are of similar size and have similar predicted membrane topologies and biochemical characteristics. Interestingly, multiple sequence alignment reveals that these oatps have 15 cysteine residues in common ( Fig. 42.1 ) that are highly conserved over the entire oatp family. It can be hypothesized that these cysteines provide an important component of transporter function through the formation of intramolecular and possibly intermolecular disulfide bonds.

Multiple sequence alignment of the oatps which are present in liver from rat, mouse, and humans was performed and revealed that there are 15 cysteine residues in common that are highly conserved over the entire oatp family, as indicated by the asterisks. Many other regions of high homology are also seen in this figure. A functional role for these regions have not as yet been elucidated. Accession numbers for the sequences in this figure are as follow: (1) oatp1a1—NM_017111; (2) Oatp1a1—NM_013797; (3) oatp1a4—NM_131906; (4) Oatp1a4—NM_030687; (5) OATP1A2—NM_021094; (6) oatp1b2—NM_031650; (7) Oatp1b2—NM_020495; (8) OATP1B1—NM_006446; (9) OATP1B3—NM_019844; (10) oatp2b1—NM_080786; (11) OATP2B1—NM_007256.

42.2.3.1

Biochemistry

Oatps in the liver are localized normally to the basolateral (sinusoidal) plasma membrane of the hepatocyte. As predicted by computer modeling, they are hydrophobic proteins, remaining membrane bound even after extraction with 0.1 M Na ₂ CO _3. On immunoblot, oatp1a1 migrates with a molecular mass of 80 kDa. Following treatment with N-glycanase, the apparent molecular mass decreases to 65 kDa, consistent with N-linked glycosylation. It has been suggested that glycosylation of oatp1a1 is a determinant of its trafficking to the plasma membrane as well as possibly its transport activity.

42.2.3.2

Function

The function of oatps, including oatp1a1, have been examined in several systems including cDNA injected Xenopus oocytes, transiently transfected cells in which transporter was functionally expressed, and in permanently transfected cells in which oatp1a1 expression is under control of a metallothionein promoter. While these studies have provided a good deal of information regarding compounds that OATPs can transport in vitro, their function in vivo has been less clear. Recently, there have been several functional studies with a targeted disruption of the gene encoding oatp1b2 in mice. This targeted disruption was not liver specific and of interest is the fact that there was no alteration of fertility, development, or viability of these mice. The effects of disruption of Oatp1b2 on its transport function were somewhat subtle. The knockout mice had an approximately fourfold reduction in the liver-to-plasma ratio of rifampicin, smaller differences (less than twofold) for cerivastatin and lovastatin, and no difference for pravastatin or simvastatin. They also had reduced plasma clearance of dibromosulfophthalein (DBSP) and were protected from the toxic effects of microcystin and phalloidin. In further studies, mice in which expression of all members of the Oatp1a and Oatp1b families were knocked out were prepared. These mice had reduced plasma clearance of a number of drugs including methotrexate and fexofenadine. They also had elevated levels of unconjugated bile acids as well as conjugated hyperbilirubinemia. There was no change in plasma levels of conjugated bile acids.

It should be noted that there is no true homolog of murine Oatp1b2 in human liver. Although OATP1B1 and OATP1B3 have been suggested as homologs of the murine protein, their amino acid sequences are only 64% and 66% identical, respectively, to that of mouse Oatp1b2. In addition, the mouse protein has a PDZ consensus-binding site at its C-terminus, while these two human proteins do not. However, in a recent large population-based study of patients taking simvastatin, 85 subjects who developed signs of myopathy, a known side effect of this drug, had a polymorphism in the gene encoding OATP1B1. Several previous smaller studies also suggested that polymorphisms in OATP1B1 were associated with altered statin pharmacokinetics. Coincidentally, the elevation of conjugated bilirubin levels in the double knockout mice is similar to that has been described in patients with the Rotor syndrome, a rare disorder in which there is chronic elevation of conjugated bilirubin in plasma with otherwise normal routine liver function tests. These individuals have recently been found to have simultaneous null mutations in the genes encoding OATP1B1 and OATP1B3. It was suggested that after conjugation with glucuronic acid in the hepatocyte, bilirubin glucuronides are effluxed from the cell into the circulation subsequently undergoing OATP-mediated reuptake. In the absence of both OATP1B1 and OATP1B3, such as that occurs in Rotor syndrome patients, levels of conjugated bilirubin in the circulation rise. It has been suggested that after the formation in the hepatocyte, bilirubin glucuronides are pumped out of the cell back into the sinusoidal circulation by the basolateral plasma membrane protein, mrp3 (AbcMRP3 (ABCC3)c3). These conjugates are then subjected to reuptake by neighboring hepatocytes, mediated by the basolateral plasma membrane proteins OATP1B1 and OATP1B3.

42.2.3.3

Regulation of Oatp Function

42.2.3.4

Transported Substrates

42.3.2.1

Biochemical Studies

Similar to the case for nonbile acid organic anions, the kinetics and characteristics of bile acid transport by hepatocytes are indicative of a carrier-mediated uptake mechanism. In an attempt to identify a hepatocyte bile acid transporter, initial studies utilized photoaffinity technology to covalently attach radiolabeled bile acid analogs to putative hepatocyte basolateral plasma membrane transporters. Two candidate transporters of 54 and 48 kDa were identified using this approach. Additional studies performed in rat liver basolateral plasma membranes utilizing radiation inactivation analysis revealed a minimal functional mass of 170 kDa for the Na ⁺ -dependent taurocholate transporter. However, none of these studies actually identified an Na ⁺ -dependent bile acid transporter that could be studied using biochemical techniques.

42.3.2.2

Molecular Biology Studies

It was not until the development of expression cloning techniques that an Na ⁺ -dependent bile acid transporter, termed the Na ⁺ -taurocholate-cotransporting polypeptide (NTCP) was identified. The NTCP cDNA that was cloned in Xenopus oocytes mediates Na ⁺ -dependent uptake of a number of bile acids. It encodes a protein with a predicted molecular mass of 39 kDa that migrates at 49 kDa on SDS-PAGE as a result of glycosylation. On deglycosylation, the protein migrates at approximately 33 kDa. In rats, expression of NTCP is limited to hepatocytes, where it resides on the sinusoidal (basolateral) plasma membrane as well as in an intracellular vesicular pool that cycles to and from the plasma membrane depending on its phosphorylation state (see below).

42.3.2.3

Physiologic Role of NTCP and the Possibility of Other Na ⁺ -Dependent Bile Acid Transporters

A physiologic role for NTCP in bile acid transport had been asserted but proof was elusive until studies performed in NTCP knockout mice showed that absence of the transporter was associated with reduced serum bile acid clearance. Subsequently, several patients have been described with elevated serum bile acid levels associated with mutations in the NTCP gene. Interestingly, NTCP has also been shown to mediate entry of hepatitis B and D viruses into hepatocytes. This is due to binding of the virus to NTCP and subsequent endocytic processing. Of note is the fact that NTCP localizes in endocytic vesicles with the epidermal growth factor receptor (EGFR) and that this association has been shown to be required for trafficking of NTCP through the cell.

42.3.2.4

Substrates of the Na ⁺ -Dependent Bile Acid Transport System

Specificity of the Na ⁺ -dependent bile acid uptake system was examined in isolated rat hepatocytes by studying the ability of 100 μM concentrations of test ligands to inhibit uptake of 5 μM ¹⁴ C-taurocholate. These studies showed no influence of hydroxyl group orientation (α or β) on ability to inhibit taurocholate uptake. In contrast, the fewer the number of hydroxyl groups on the steroid ring, the more potent the inhibition while substitution of hydroxyl groups by keto groups resulted in the reduced inhibition of taurocholate uptake. Side chain length was also a factor in inhibitory potency, with increased inhibition of taurocholate uptake with side chains up to 11 Å and reducing inhibition with longer side chains. As inhibition of taurocholate uptake by these compounds was competitive, the assumption is that they represent substrates for the hepatocyte Na ⁺ -dependent bile acid uptake system, although this may not necessarily be the case. Further studies examined the inhibition of uptake of ¹⁴ C-taurocholate into isolated rat hepatocytes by a series of trihydroxy bile acids with differing side chain length and charge. These studies found that long side chains (> 8 Å) with no or negative charge were potent inhibitors of taurocholate uptake. Positive charge on the side chain or shortening of the side chain reduced inhibitory potency.

More limited studies of the substrate specificity of NTCP have been performed. In general, for rat NTCP, there was modest relationship of NTCP-mediated transport to substrate structure. Conjugated bile acids were transported better than unconjugated bile acids and there was little influence on uptake of the configuration of the hydroxyl groups (α or β) on the steroid ring. Interestingly, rat NTCP mediated high-affinity uptake of BSP, a nonbile acid organic anion as well the 3-O-sulfate conjugate of 17α-ethinylestradiol. Substrate specificities were also examined for rabbit NTCP and the rabbit Na ⁺ -dependent ileal bile acid transporter. A substrate activity model was devised for the ileal transporter, but a similar model does not yet exist for rabbit NTCP. It should be noted that as compared to the rat and human NTCP counterparts, rabbit NTCP is only 75% and 82% identical, respectively. This might explain differences in substrate specificities that were reported for rat and rabbit NTCPs. Applicability of these studies to human NTCP has not been clear. More recent studies examined substrate requirements for transport by human NTCP. Pharmacophore analysis revealed that the steroidal hydroxyl groups and C-24 steric interaction play a key role in NTCP binding and transport of substrates. The model based on these studies was used to identify potential NTCP substrates from a database of FDA approved drugs, resulting in the identification of irbesartan and losartan as novel NTCP ligands.

42.3.3.1

Phylogeny

Na ⁺ -dependent bile acid uptake by the liver is not seen in phylogenetically old organisms such as the skate, although they are able to take up bile acids by Na ⁺ -independent means. The Na ⁺ -independent bile acid transport was expressed in skate liver mRNA injected X. laevis oocytes, and subsequently a skate homolog belonging to the oatp family was cloned. Other studies identified another novel protein (organic solute transporter, OST) in skate and mammalian liver that can transport a number of organic solutes including taurocholate. The OST is a heterodimer of OSTα and OSTβ subunits. In mammals, OST is abundant in the intestine and is upregulated in the liver during cholestasis where it may serve in a protective role to allow egress of toxic bile acids across the hepatocyte basolateral membrane into the circulation.

42.3.3.2

Ontogeny

Bile acid transport has also been studied during development. The V _max for initial Na ⁺ -dependent uptake of taurocholate by basolateral liver plasma membrane vesicles prepared from day 22 fetal, day 1, and day 14 neonates was, respectively, 23%, 36%, and 47% of that seen in basolateral liver plasma membrane vesicles prepared from adults. Interestingly, NTCP mRNA levels in rat liver are less than 20% of adult levels prior to birth, increasing to 35% of adult levels by the first day after birth, and achieving adult levels by 1 week of age. The NTCP protein was approximately 8% of adult levels just prior to birth, increasing to 82% of adult levels at 1 day after birth. However, the size of the protein was approximately 39 kDa until 4 weeks of age when it migrated at the approximately 49 kDa size seen in adults. This difference in apparent size was due to differences in glycosylation. This dissociation of total liver NTCP expression from Na ⁺ -dependent bile acid transport is unexplained, but could be due in part to the presence of other Na ⁺ -dependent bile acid transporters with different developmental patterns. Although differences in subcellular targeting could explain these differences, plasma membrane localization of the transporter had an adult appearance by 5 days after birth.

42.3.3.3

Liver Regeneration

As is the case for nonbile acid organic anions described above, Na ⁺ -dependent uptake of taurocholic acid is markedly reduced within 24 hours following two-thirds partial hepatectomy in the rat. Serum bile acid levels are substantially increased for at least 1 week as compared to sham-operated controls. Expression of NTCP protein is reduced by over 90% at 24 hours of partial hepatectomy, and normalizes by approximately 1 week.

42.3.3.4

Altered Subcellular Distribution of Transporter

Studies in rat hepatocytes showed that maximal Na ⁺ -dependent taurocholic acid transport rate was increased within minutes by approximately 50% following pretreatment with cAMP. This increase in taurocholate transport was associated with translocation of NTCP from an intracellular pool to the plasma membrane. This phenomenon was explained by the finding that NTCP exists within the hepatocyte in a phosphorylated form and that it undergoes dephosphorylation in response to cAMP, subsequently translocating to the plasma membrane. This cAMP-dependent dephosphorylation event appears to be mediated by protein phosphatase 2B (PP2B).

The mechanisms that mediate NTCP trafficking through the cell are complex and in addition to the factors noted above, utilize a phosphatidylinositol-3-kinase-dependent pathway that may interact with actin filaments as well as microtubules. Studies of NTCP-transfected cells have helped to elucidate mechanisms of NTCP trafficking. HepG2 cells that had been transiently transfected with GFP-NTCP had a 40% increase in GFP fluorescence at the plasma membrane within 2.5 minutes of incubation in cAMP. Cytochalasin D that disrupts the actin-based microfilament cytoskeleton blocked this effect. Nocodazole, a disruptor of microtubules, prevented GFP-NTCP from reaching the plasma membrane even under basal conditions. This effect was reversible within 2 hours after the removal of nocodazole. These experiments are consistent with the hypothesis that targeting of NTCP to the plasma membrane is a two-step process in which the first step involves delivery of NTCP to a subplasma membrane location via microtubules; following which NTCP transfers to microfilaments targeting it to the plasma membrane. Further studies were performed in a cell-free system in which motility of NTCP-containing intracellular vesicles could be examined. In these studies, a microassay chamber was used in which the glass surface was coated with fluorescent microtubules. Vesicles were flowed into this chamber and antibody against NTCP followed by fluorescent secondary antibody were added. These studies revealed that NTCP-containing vesicles attached to microtubules and moved upon addition of ATP. Immunofluorescence analysis showed that the minus-end directed microtubule motor dynein, and the plus-end directed microtubule motors kinesin-1 and kinesin-2 were associated with these vesicles. Consistent with previous studies that suggested that trafficking of NTCP is regulated at least in part by the PI3 kinase/PKCζ pathway, addition of a PKCζ pseudosubstrate, a potent inhibitor of PKCζ, virtually eliminated microtubule-based vesicle motility. Similar results were seen in HuH7 cells expressing GFP-NTCP following the addition of a cell permeant form of the PKCζ pseudosubstrate. To characterize the PKCζ target of phosphorylation, NTCP-containing endocytic vesicles were incubated with γ- ³² P-ATP, revealing a 180 kDa phosphoglycoprotein that was identified as the EGFR. The quantification of biotinylated NTCP following surface biotinylation of HuH7 cells expressing green fluorescent protein (GFP)-NTCP revealed substantially reduced trafficking of NTCP to the cell surface with EGFR knockdown. In addition, microtubule-based motility of NTCP-containing endocytic vesicles was also significantly reduced when they were not associated with EGFR. When cells were incubated with EGF, a fraction of NTCP internalized. These results suggest a model in which NTCP and EGF-EGFR internalize into common endocytic vesicles from which they segregate, trafficking EGF-EGFR to lysosomes and recycling NTCP to the plasma membrane. Regulation of NTCP trafficking by EGF may be important in subcellular targeting of NTCP ligands such as hepatitis B.

42.3.3.5

Sex Hormones and Pregnancy

The Na ⁺ -dependent uptake of taurocholate is higher in rat hepatocytes obtained from males compared with females. This correlates with the finding that NTCP is reduced in basolateral plasma membrane fractions prepared from female as compared to male rats. Of note is the fact that lipid fluidity was also decreased in plasma membrane fractions prepared from females as compared to males. Interestingly, oatp1a1 levels were unchanged as distinct from findings in rat kidney. Further studies showed that this sexually dimorphic expression of NTCP was regulated by a number of hormones, including estrogens. Hormonal regulation of bile acid uptake was seen by reduction in uptake of taurocholate in hepatocytes isolated from pregnant as compared to nonpregnant rats. Two days postpartum, Na ⁺ -dependent uptake of taurocholate is increased to levels above those seen in nonpregnant female rats in both hepatocytes and basolateral plasma membrane vesicles prepared from female rats. In large part this overshoot in Na ⁺ -dependent taurocholate uptake is regulated by prolactin. Whether NTCP expression correlates with altered transport of taurocholate in pregnancy remains unclear, as in one study using basolateral plasma membrane vesicles from pregnant rats, there was no change in NTCP content while in another study, NTCP content in plasma membrane fractions prepared from pregnant rat liver was reduced by 60%. Whether these differences were due to differences in membrane preparations and subcellular distribution of NTCP remains to be elucidated. In a study using mouse liver homogenates, NTCP protein and mRNA were found to be reduced during pregnancy.

42.3.3.6

Cholestasis

Hepatocyte bile acid uptake transporters are downregulated in cholestasis. For example, following common bile duct ligation in the rat, NTCP protein and mRNA expression are profoundly reduced. However, a number of studies suggest that bile acid uptake following common bile duct ligation is relatively well maintained. In one study, rats were subjected to 5 days of common bile duct obstruction following which obstruction was removed. Serum bile acids, which had been highly elevated, returned to normal within 60–90 minutes of relief of obstruction. This was accompanied by prompt excretion of bile acids into bile. Plasma clearance of a tracer dose of taurocholate injected at the time of relief of biliary obstruction was near normal. This normalization of transport function would be expected to occur faster than the return of NTCP levels to normal. In another study, hepatocytes were isolated from rats in which the common bile duct had been ligated for 7 days. The NTCP protein levels were reduced by 90% while the V _max for Na ⁺ -dependent taurocholate uptake was reduced by only 70%. When basolateral plasma membrane vesicles were prepared from livers of rats in which the common bile duct was ligated for 50 hours, Na ⁺ -dependent taurocholate uptake remained normal. Transport studies were also performed in isolated perfused livers from rats 24 and 72 hours following common bile duct ligation. Quantitation of bile acid uptake following addition of ³ H-taurodeoxycholate to the perfusate (16 nmol/min/g) showed little if any difference in uptake between control and bile duct ligated livers. Perfusion with a larger dose of this bile acid (4000 nmol/min/g) resulted in an approximately 35% reduction in maximal uptake by livers that had undergone common bile duct obstruction for either 24 or 72 hours. In the aggregate, these studies show at most a modest reduction of hepatocyte bile acid uptake following mechanical cholestasis in the face of a profound reduction in NTCP protein and mRNA. These data suggest the possibility of other Na ⁺ -dependent bile acid transporters in addition to NTCP. They also point out the potential importance of upregulation of bile acid efflux pumps on the sinusoidal plasma membrane (e.g., MRP3 and MRP4) as a mechanism to protect the hepatocyte from toxic concentrations of bile salts during cholestasis.

42.4.2.1

Studies in Purified Bile Canalicular Plasma Membrane Vesicles

This field was stimulated greatly by the ability to quantify transport of radiolabeled ligands into purified preparations of inside-out rat liver bile canalicular plasma membrane vesicles. Several studies demonstrated ATP-dependent uptake of taurocholate. Affinity labeling of these membranes with a photolabile bile salt derivative identified a 110 kDa glycoprotein. The ATP-dependent taurocholate transport was reconstituted in proteoliposomes containing this protein and it was presented as the ATP-dependent bile acid transporter. Purification and sequencing of this 110 kDa protein identified it as a known rat bile canalicular ecto-ATPase. Transfection of COS cells with the corresponding cDNA was reported as conferring ATP-dependent efflux of taurocholate. However, subsequent studies found that ATP-dependent bile acid transport was present in a subpopulation of canalicular membrane vesicles that had little ecto-ATPase activity. Similar findings were reported in liver cell lines that retained ATP-dependent bile acid excretion but had little ecto-ATPase expression.

42.4.2.2

Identification of the Bile Canalicular Bile Acid Excretory Pump

It was later shown that the 160 kDa bile canalicular protein originally named sister of P-glycoprotein (spgp) conferred ATP-dependent taurocholate transport in cRNA injected X. laevis oocytes as well as in membrane vesicles purified from Sf9 cells that had been transfected with a plasmid encoding spgp. This protein has now been renamed bile salt export pump (bsep) and the gene has been designated as Abcb11 . That bsep is physiologically important has been demonstrated by finding that it is mutated in patients with the inheritable disorder progressive familial intrahepatic cholestasis type 2 (PFIC2). This disorder is associated with very high levels of bile acids in serum and low levels in bile. Targeted inactivation of this gene in mice resulted in a much less severe phenotypic picture. Although secretion of cholic acid was greatly reduced (6% of wild type) in mutant mice, total bile salt excretion was somewhat conserved (30% of wild type). This secretion was accounted for by increased excretion of tetra-hydroxylated bile acids, which were not detected in wild-type mice. These results suggest that at least in the mouse, there may be alternative bile canalicular transporters that can mediate excretion of hydrophilic bile acids.

The substrate specificity of bsep has not been examined in great detail, although it has been shown to mediate transport of the bile acids taurocholate, glycocholate, taurochenodeoxycholate, glycochenodeoxycholate, and tauroursodeoxycholate. In addition, a number of nonbile acid organic anionic compounds have been shown to be competitive inhibitors of bsep-mediated taurocholate transport. These compounds include rifampicin, cyclosporine, and glibenclamide. Whether these compounds are themselves substrates for bsep has not as yet been established directly, although it has been suggested that some instances of drug-induced cholestasis may result from direct inhibition of bsep transport activity. Interestingly, pravastatin, but not other statins, is a transported substrate. Pharmacophore modeling may be of help in predicting drugs that may inhibit BSEP function, leading to cholestasis.

42.4.2.3

Identification of Other Proteins that Influence Bile Canalicular Excretion of Bile Acids

Study of patients with two additional phenotypically similar forms of progressive familial intrahepatic cholestasis has yielded exciting insights into additional mechanisms of bile canalicular transport function. PFIC1 has been found to be due to the mutation of the gene encoding a P-type ATPase, named Fic1 (ATP8B1), which functions as an aminophospholipid translocator. It is present in greatest concentration in the intestine but is also present in the liver where it has been localized to the bile canalicular membrane. The relationship of this transport protein to abnormal bile acid excretion remains to be elucidated, although recent evidence suggests that deficiency of Fic1 is associated with enhanced bile acid extraction of cholesterol from the bile canalicular membrane. This is hypothesized to reduce the transport activity of bsep, leading to cholestasis.

PFIC3 has been found to be due to the mutation of the gene encoding MDR3(ABCB4), a bile canalicular membrane flippase that is thought to move phospholipids from the inner leaflet of the canalicular membrane to the outer leaflet, where it can form micelles with cholesterol and bile acids. Affected patients have low levels of phospholipids in bile. Interestingly, knockout mice in which expression of mdr2, the mouse homologue of human MDR3, is disrupted have a similar type of liver disease that coincides with inability of the liver to secrete phospholipids into the bile. Canalicular bile acid excretion is normal in mdr2/MDR3 deficiency, and it is believed that the fact that bile acid monomers are not associated with phospholipid in micelles may result in direct toxicity to cholangiocytes and hepatocytes.

42.4.2.4

Identification of Bile Canalicular Proteins That Mediate Excretion of Nonbile Acid Organic Anions

Nonbile acid organic anions are also excreted across the bile canaliculus. The mechanism by which bilirubin glucuronides are excreted has been of long-standing interest, in light of the fact that when this process is disrupted in disease, patients develop jaundice and excrete these bilirubin conjugates into urine. The existence of an ATP-dependent canalicular export pump for bilirubin glucuronides was first observed in studies of bilirubin glucuronide transport by rat liver canalicular membrane vesicles. Of note is the fact that this transport was deficient in vesicles prepared from TR(−) rat livers. These rats represent an animal model of the Dubin-Johnson syndrome, an inheritable disorder characterized by defective excretion of bilirubin glucuronides but normal excretion of bile acids. Subsequent studies revealed that an isoform of the multidrug resistance protein (MRP) was absent from the canalicular membrane of the TR(−) rats and patients with the Dubin-Johnson syndrome. This bile canalicular MRP has been termed MRP2(ABCC2). This 190 kDa protein is expressed predominantly in hepatocytes and its amino acid sequence is 49% identical to that of MRP1, a protein with broad distribution. It should be noted that although mrp2 expression is absent in TR(−) rats, in vivo excretion of endogenous bilirubin conjugates is reduced by only 40%. Infusion of a bilirubin load in vivo or in isolated perfused liver revealed a 98% reduction in conjugated bilirubin excretion into bile. These results suggest the existence of other, as yet unidentified, canalicular transporters that have a low capacity for transport of bilirubin conjugates. Unexpectedly, mice in which expression of the actin scaffolding protein, radixin, had been knocked out were found to have markedly reduced expression of mrp2 at the canalicular plasma membrane, although total cell content of the protein was reduced by only 40%. Presumably, radixin serves as a chaperone that directly or indirectly facilitates the trafficking of mrp2 to the apical plasma membrane of hepatocytes. This trafficking requires C-terminal 15 amino acids of mrp2 and is unaffected by the removal of the C-terminal 3 amino acids that would be essential for interaction with a PDZ domain-containing protein.

Mrp2 has a broad substrate specificity. It may also represent an alternative transporter for some bile acids, as it can mediate excretion of 3-glucuronide and 3-sulfate dianionic bile acids and ester sulfate conjugates of lithocholic acid. Taurocholate is not a substrate for mrp2, but it is transported by mrp3, a protein that is related to mrp2 but is localized to the hepatocyte sinusoidal (basolateral) plasma membrane. Expression of mrp3 on the basolateral membrane is highly increased in TR(−) rats, in patients with the Dubin-Johnson syndrome, and in cholestasis. It has been suggested that it may serve as a compensatory mechanism for solute elimination from the hepatocyte when canalicular secretion is blocked. Other members of the mrp family have also been shown to increase in the liver during cholestasis. Their physiologic roles in health and disease remain to be elucidated.

42.4.3.1

Phylogeny and Ontogeny

It is of interest that hepatic excretion of organic anions has been highly conserved during vertebrate evolution and homologues of mrp2 and bsep have been described in the liver of the skate ( Raja erinacea ), a 200 million year old vertebrate. During liver regeneration in the rat following two-thirds partial hepatectomy, there is no change in protein expression of these transporters, although ontogenic studies in the rat demonstrated that adult levels were not attained until at least 4 weeks of age. Several studies in rats showed that mRNAs encoding these proteins were near adult levels early in life and correlated poorly with actual protein expression. Another study in rats showed significantly decreased hepatic mrp2 mRNA levels and increased bsep mRNA levels through day 26 as compared to day 42. Yet another study in rats revealed that levels of both these proteins in liver were near adult levels by 1 week of age. The reasons for these differences are not clear at this time. Functionally, biliary excretion of substrates for these transporters is reduced in the days after birth, although there is little information regarding the time course of maturation of this process. A study performed in human fetal liver showed modestly reduced mRNA levels of these transporters compared with adult liver without statistical significance, in part reflecting a large variability in results.

42.4.3.2

Pregnancy

When expressed per gram of liver tissue, maximal biliary excretion of bile acids and other organic anions is reduced in liver from pregnant rats. As the liver is larger during pregnancy, when expressed per whole liver, there is little difference in maximal excretion of these compounds. When mrp2 was quantified in liver homogenates prepared from pregnant rats, it was found to be decreased by approximately 50% when equal amounts of total liver homogenate protein were compared. The quantification of bsep in a mixed liver plasma membrane preparation showed no difference between pregnant and nonpregnant rats, while mrp2 was again found to be reduced by approximately 50%. Postpartum, bsep concentration in liver was found to increase by as much as 70%, while there was little change in mrp2 concentration. In isolated perfused livers obtained from postpartum rats, maximal secretory capacity for taurocholate increased by as much as 30%. These effects on bsep and mrp2 protein expression could be reproduced by treating ovariectomized rats with prolactin for 1 week.

42.4.3.3

Cholestasis

Cholestasis can be defined as a syndrome resulting from impaired excretion of bile acids and impaired bile formation. Cholestasis may result from altered expression of specific transporters, as detailed above, or from mechanical or metabolic causes. Studies performed in cholestasis resulting from treatment of rats with endotoxin revealed marked reduction of bsep and mrp2 protein levels in membrane fractions within 15 hours. Immunofluorescence examination revealed a “fuzzy” pattern along the canalicular membrane consistent with the possibility of redistribution of these transporters to a subapical vesicular compartment. Following 3 days of mechanical obstruction of the common bile duct in rats, bsep protein in purified plasma membrane fractions was reduced by approximately 40% while mrp2 was reduced by approximately 80%. Similar findings were obtained for cholestasis resulting from treatment with ethinylestradiol or endotoxin. Although subcellular distribution of bsep, as assessed by immunofluorescence, appeared to be normal in all the three models of cholestasis, relocalization of transporters to a vesicular compartment adjacent to the bile canalicular membrane could not be ruled out by these studies. Such intracellular relocalization resulting in functional inactivation could account for the substantial reduction in bile salt output in rats in which bile duct integrity was restored by the insertion of a catheter 14 days after common bile duct ligation. Clear demonstration of intracellular redistribution of mrp2 was described in rat models of intrahepatic and obstructive cholestasis. These studies emphasize the importance of assessing both protein content as well as subcellular distribution as a means to better understand cellular function.

42.4.3.4

Transporter Trafficking

Mechanisms for the trafficking of bile canalicular membrane proteins between intracellular and surface locations have been the subject of recent study. Initial experiments indicated that infusion of dibutyryl cAMP or taurocholic acid into an isolated perfused rat liver resulted in increased bile secretion. Later studies revealed that treatment with these agents results in the recruitment of transporters from intracellular stores to the canalicular membrane and that this process requires intact microtubules and is regulated by PI 3-kinase. The effects of cAMP and taurocholate on transporter recruitment to the canalicular plasma membrane appear to be independent, suggesting the existence of two intracellular pools of canalicular transporters. Whether these compartments are related to the subapical compartment and apical endocytic vesicle compartments that have been described in the hepatic WIF-B cell line remains to be determined. Recent studies in which yellow fluorescent protein (YFP) tagged bsep was expressed in the polarized WIF-B9 liver cell line revealed abundant intracellular expression of this protein in tubulo-vesicular structures that also contained Rab11, a marker of recycling endosomes. Exchange of YFP-bsep between the canalicular plasma membrane and these intracellular structures was inhibited by colchicine, a microtubule disruptor. However, unlike findings in intact liver, there was no effect of cAMP, taurocholate, or PI 3-kinase inhibitors on this trafficking. The mechanistic basis for vesicular trafficking of canalicular transporters is not well understood. As noted above, the scaffolding protein radixin is required for bile canalicular localization of mrp2. Whether this is related to the finding that apical targeting of mrp2 requires the presence of its carboxyl-terminal 15 amino acids is not known. HAX-1, a 34 kDa protein, that can link proteins to cortactin has been found to associate with bsep and mdr2, and appears to be required for their internalization but not delivery to the apical plasma membrane. The mechanism by which cortactin, HAX-1, and canalicular transporters interact remains to be elucidated.

42.4.3.5

Transcriptional Regulation

Recognition in 1999 that bile acids were endogenous ligands for the farnesoid X receptor (FXR) resulted in new insights regarding regulatory mechanisms for transporter expression. Both unconjugated as well as glycine- and taurine-conjugated bile acids can serve as ligands for FXR. Mrp2 and bsep are included in the list of target genes whose transcription is upregulated by bile acid bound FXR. The FXR-response elements have been shown to reside in the genes encoding these transporters. Transcription of the orphan nuclear receptor known as small heterodimer partner (SHP) is also increased by FXR activation, and interaction of SHP with the orphan nuclear receptor LRH-1 results in the repression of the gene encoding Cyp7A1, a rate-limiting bile acid biosynthetic enzyme, thereby reducing bile acid synthesis. Expression of the gene encoding Cyp8B1, another bile acid biosynthetic enzyme, is also repressed due to interaction of SHP with LRH-1 and HNF-4α. SHP has also been described as suppressing the promoter for rat NTCP. In these ways, FXR is the lynchpin in an exquisitely sensitive system that coordinates bile acid synthesis and excretion. This is emphasized by the finding of severe hepatotoxicity following cholic acid feeding to FXR knockout mice. Interestingly, although steady-state mRNA levels for bsep are reduced in these mice, steady-state levels of mRNA for mrp2 and NTCP are normal. There have been no reports as yet regarding transporter protein levels or trafficking in these mice.

In addition to FXR, the nuclear receptor PXR also regulates transcription of several transporter genes. Its ligands include bile acids and organic anions such as rifampicin. It has been described as increasing the mRNA levels of rat, mouse, and human MRP2, mouse oatp1a4, mouse bsep, mouse mrp3, and rat oatp1a4. Differences in the amino acid sequence of PXR have been related to species-specific responses to particular ligands. Although there is no evidence that human OATP1B1 is regulated by PXR, this transporter appears to play an important role in facilitating entry into the cell of PXR ligands such as rifampicin. Several allelic variations of the OATP1B1 gene that are found in European and African Americans have markedly reduced ability to mediate uptake of rifampicin. Whether PXR-mediated transporter function is altered in these individuals remains to be determined. Interestingly, FXR and PXR upregulation may be protective against hepatocyte toxicity resulting from cholestasis. As these nuclear hormone receptors mediate many complex events within the cell, elucidation of specific genes that ameliorate toxic events will be of great potential importance in designing new classes of pharmaceuticals.