I. NORMAL PHYSIOLOGY OF BILE SALT TRANSPORT

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Bile is a vital secretion, essential for intestinal digestion and absorption of lipids. Moreover, bile is an important route of elimination for environmental toxins, carcinogens, drugs, and their metabolites (xenobiotics). Bile is also the major route of excretion for endogenous compounds and metabolic products (endobiotics) such as cholesterol, bilirubin, and hormones (44, 262). Bile is primarily secreted by hepatocytes into minute channels arranged as a meshwork of tubules or canaliculi between adjacent hepatocytes. "Canalicular bile" accounts for ~75% of daily bile production in humans and is modified by secretory and absorptive processes as it passes along the bile ductules and ducts. The quantity of "ductular/ductal bile" varies with the species and responses to enteric hormonal stimuli and varies from as little as 5% in rats to as much as 25-40% of secretion in humans. Bile is further concentrated up to 10-fold in the gallbladder before reaching the intestine except for species like the rat where the gallbladder is absent (44, 262).

Bile secretion is an osmotic process driven predominantly by active excretion of organic solutes into the bile canaliculus, followed by passive inflow of water, electrolytes, and nonelectrolytes (e.g., glucose) from hepatocytes and across semipermeable tight junctions (42, 332). The main organic solutes of bile are bile salts, phospholipids, and cholesterol, which form mixed micelles in bile. The vectorial excretion of bile salts from blood into bile represents the major driving force for bile flow ("bile salt-dependent" bile flow). The human bile salt pool size is ~50-60 µmol/kg body wt and averages 3-4 g. Most of the bile salt pool is stored in the gallbladder during the fasting state (139, 210, 245). Canalicular excretion of reduced glutathione (GSH) and bicarbonate (HCO) constitute the major components of the "bile salt-independent" fraction of bile flow. However, HCO secretion occurs mainly at the level of bile duct epithelial cells (cholangiocytes), in response to stimulation by hormones and neuropeptides such as secretin, vasoactive intestinal peptide, bombesin, etc. (22, 39, 167).

Many organic biliary constituents including bile salts, cholesterol, and phospholipids are reabsorbed with high efficiency after reaching the small intestine and recirculate via the portal blood back to the liver. Thus the human bile salt pool circulates 6-10 times per day, resulting in a daily bile salt excretion of 20-40 g. Despite a high degree of intestinal bile salt conservation, 0.5 g of bile salts are lost through fecal excretion and must be replaced by de novo bile salt synthesis, which thus contributes to only 3-5% of the bile salts that are excreted into bile (139, 210, 245).

Bile salts may also undergo "cholehepatic shunting" from the bile duct lumen, via cholangiocytes and the periductular capillary plexus. Bile salt reabsorption by cholangiocytes may contribute in part to the conservation of bile salts and the generation of a hypercholeretic bile flow (123, 395). Although this pathway probably plays a minor role under normal physiological conditions, cholehepatic shunting of bile salts may become an important escape route for bile salts under cholestatic conditions when the bile duct epithelium proliferates. Moreover, bile salt uptake into cholangiocytes and gallbladder epithelial cells may also play an important role for cell signaling in the regulation of secretory and proliferative events within the biliary tree (7, 10, 67).

Bile salts are efficiently removed from portal blood by the liver via high-affinity, low Michaelis constant (K_m) transporting polypeptides in the basolateral sinusoidal membrane. Bile salts that escape the first-pass clearance by the liver are filtered at the glomerulus and excreted into urine, where they are reabsorbed by transporters in the brush border of the proximal convoluted tubule (386).

Most hepatic transport systems are not fully expressed until shortly before/after birth (19). Thus the fetus must rely entirely on elimination of "biliary" constituents via the placenta and maternal liver. This "placenta-maternal liver tandem" is responsible for protecting the fetal organism from potentially harmful compounds.

Recent cloning studies have characterized the molecular properties of most of the hepatobiliary transport systems required for uptake and excretion of bile salts and other biliary constituents in liver and extrahepatic tissues (Table 1, Fig. 1). These studies have done much to advance our understanding of the molecular basis of bile formation (205, 210, 245, 258, 259, 342, 358, 359). However, bile formation normally depends not only on the proper function of these transport systems, but also on an intact cytoskeleton required for the movement of vesicles and bile canalicular contractions, and on junctional contacts that seal off the bile canaliculi and maintain cell polarity. Signal transduction mechanisms also regulate and coordinate these various processes. Although this review is focused on the molecular mechanisms involved in bile salt transport in the liver and the enterohepatic circulation, the interested reader may wish to consult other reviews that address these cellular mechanisms (15, 44, 98, 250, 251, 286).

View larger version (36K): [in this window] [in a new window]   Fig. 1. Hepatobiliary transport systems in liver and extrahepatic tissues in humans. Bile salts (BS) are taken up by hepatocytes via the basolateral Na+/taurocholate cotransporter (NTCP) and organic anion transporting proteins (OATPs). Monovalent BS are excreted via the canalicular bile salt export pump (BSEP) while divalent BS together with anionic conjugates (OA) are excreted via the canalicular conjugate export pump (MRP2). The phospholipid export pump (MDR3) facilitates excretion of phosphatidylcholine (PC), which forms mixed micelles in bile together with BS and cholesterol. Cationic drugs (OC+) are excreted by the multidrug export pump (MDR1). Other basolateral isoforms of the multidrug resistance-associated protein (MRP1 and MRP3) provide an alternative route for the elimination of BS and nonbile salt OA from hepatocytes into the systemic circulation. BS are reabsorbed in the terminal ileum via ileal Na+-dependent bile salt transporter (ISBT) and effluxed by MRP3. Similar mechanisms exist in proximal renal tubules and cholangiocytes where an additional, truncated isoform (t-ISBT) may be involve in BS efflux from cholangiocytes. MRP2 is also present in the apical membrane of enterocytes and proximal renal tubules, while MDR1 is also found in intestine and bile ducts.

The hepatocyte is a polarized epithelial cell with basolateral (sinusoidal) and apical (canalicular) plasma membrane domains. Bile salts are concentrated in bile by active transport systems that are arranged in a polarized fashion (Table 1, Fig. 1). Hepatic uptake of biliary constituents (and their precursors) is initiated at the basolateral membrane, which is in direct contact with portal blood plasma via the fenestrae of the sinusoidal endothelial cells and the space of Disse. After uptake into hepatocytes, bile salts and other cholephiles reach the canalicular pole by diffusion either in the aqueous cytoplasm or within intracellular lipid membranes, depending on their hydrophobicity. The canalicular membrane represents the excretory pole of the hepatocyte and forms the border of the bile canaliculus. Canalicular excretion of biliary constituents represents the rate-limiting step of bile secretion since biliary constituents are excreted against high concentration gradients into bile. The basolateral and canalicular membranes differ in their biochemical composition and functional characteristics and are separated by tight junctions that seal off the bile canaliculi and hence form the only anatomical barrier maintaining the concentration gradients between blood and bile (38, 44).

Basolateral bile salt transport systems are essential for bile formation since ~95% of bile salts excreted into bile by the liver are reabsorbed on each pass through the intestine and undergo an "enterohepatic circulation" (139). This process is highly efficient with first-pass extraction rates of conjugated bile salts in the range of 75-90% depending on bile salt structure and is independent of systemic bile salt concentrations (241). Unconjugated bile salts are weak acids that are uncharged at the physiological pH in plasma and thus can traverse cell membranes by passive diffusion. However, taurine or glycine conjugated bile acids require an active transport mechanism for cellular uptake (241). Hepatic uptake of bile salts occurs against a 5- to 10-fold concentration gradient between the portal blood plasma and the hepatocyte cytosol and is mediated by both sodium-dependent and -independent mechanisms (262). Most substances cleared by the liver (including bile salts) are highly protein bound to serum albumin. Because only the unbound fraction of biliary constituents enter the liver, the ligand must dissociate upon contact with the sinusoidal membrane of the hepatoctye. Therefore, efficient extraction from albumin is an important step (293). Because simple dissociation from serum albumin is too slow (100, 382), this dissociation must be facilitated at the cell membrane, although proof for an hepatocellular albumin receptor is lacking (176, 271, 383). Under physiological conditions, bile salts are removed from sinusoidal blood predominantly by zone 1 (periportal) hepatocytes (119, 159). Hepatocytes in zone 3 downstream are only recruited at higher bile salt loads (215), such as may occur postprandially or under cholestatic conditions. Thus the normal liver has considerable functional reserve capacity for bile salt uptake. There are several different transport systems involved in hepatic bile salt uptake, and these include a high-affinity Na⁺-dependent bile salt transporter Ntcp/NTCP (Slc10a1/SLC10A1) and a family of multispecific organic anion transporters (Oatps/OATPs; Slc21a/SLC21A) that can mediate Na⁺-independent bile salt uptake.

A) Sodium-dependent uptake via Ntcp/NTCP. The Na⁺-dependent pathway accounts for >80% of conjugated taurocholate uptake but <50% of unconjugated cholate uptake (210, 245). Because most bile salts are conjugated, the Na⁺-dependent transport system Ntcp/NTCP is the most relevant Na⁺-dependent bile salt uptake system. Bile salt uptake via Ntcp/NTCP is unidirectional with a sodium-to-taurocholate stoichiometry of 2:1, i.e., cotransport of two Na⁺ with one taurocholate molecule, and electrogenic, i.e., it is driven by both the transmembrane Na⁺ gradient which in turn is maintained by a Na⁺-K⁺-ATPase and the intracellular electrical potential derived from the outward diffusions of K⁺ (210, 245).

The basolateral Na⁺-dependent bile salt transporter, the Na⁺-taurocholate cotransporting polypeptide (Ntcp/NTCP), has been cloned from rat, human, mouse, and rabbit liver (57, 125, 127, 198). The rat liver Ntcp (gene symbol Slc10a1) consists of 362 amino acids with an apparent molecular mass of 51 kDa, 2 NH₂-terminal glycosylation sites, and 7 putative transmembrane domains (14, 127, 337), the latter being unique among sodium cotransporters (389). The Ntcp gene is located on rat chromosome 6q24 and spans 16.5 kb, with five exons separated by four introns (210, 245). Ntcp is localized exclusively at the basolateral membrane of differentiated mammalian hepatocytes and is hepatocyte specific (14, 337). Isolation of hepatocytes results in rapid reduction of Ntcp expression and loss of hepatocellular bile salt uptake in vitro (295). Ntcp is distributed homogeneously throughout the liver lobule/acinus (337). During rat development, Ntcp can first be detected between days 18 and 21 of gestation (43). Functionally, Ntcp transports all physiological bile salts (e.g., as taurocholate, glycocholate, taurochenodeoxycholate, tauroursodeoxycholate) when expressed in oocytes and various cell systems, although its transport activity is highest for glycine- and taurine-conjugated dihydroxy and trihydroxy bile salts (210, 242, 245). Antisense experiments in Xenopus leavis oocytes injected with total rat liver mRNA have revealed a 95% reduction of Na⁺-dependent taurocholate transport (126), suggesting that Ntcp is the major if not the only Na⁺-dependent bile salt uptake system in rat liver. Although bile salts are the major physiological substrate for Ntcp, other compounds such as estrogen conjugates (estrone-3-sulfate), bromosulfophthalein, dehydroepiandrosterone sulfate, thyroid hormones, and the drug ONO-1301 can also be transported (210, 242, 245, 349). In addition, Ntcp also mediates uptake of drugs that are covalently bound to taurocholate such as chlorambucil-taurocholate (206).

In mouse liver, two alternatively spliced Ntcp isoforms (Ntcp1, Slc10a1; Ntcp2, Slc10a2) have been cloned. Ntcp is a 362-amino acid protein, while Ntcp2 is a truncated 317-amino acid protein produced by alternative splicing (57). Both Ntcps mediate Na⁺-dependent taurocholate uptake when expressed in oocytes. However, mRNA levels of Ntcp2 are ~50-fold lower than Ntcp1, and its functional relevance under normal and pathological conditions remains to be determined (57).

Human NTCP (SLC10A1) consists of 349 amino acids and shares 77% amino acid identity with rat liver Ntcp. The functional properties of NTCP are very similar to rat Ntcp, although it has a higher affinity for taurocholate than the rat and mouse orthologs (125). The NTCP gene has been mapped to chromosome 14q24.1-24.2 (210, 245).

Microsomal epoxide hydrolase (mEH) has also been proposed to mediate Na⁺-dependent bile salt uptake (365, 366, 399), including a microsomal isoform and an isoform localized to the basolateral membrane of hepatocytes. Expression of the cell-surface isoform of mEH in Madin-Darby canine kidney (MDCK) cells transfers/mediates Na⁺-dependent DIDS-inhibitable taurocholate transport (366). Although mEH could mediate Na⁺-dependent cholate uptake in hepatocytes, it has to be considered that >50% of cholate uptake is Na⁺ independent, and Ntcp also contributes to Na⁺-dependent cholate uptake (210). Moreover, mEH (/) mice have no abnormalities in bile salt homeostasis, suggesting that mEH is not an important determinant for physiological bile salt transport (253). The microsomal isoform of mEH could be involved in vesicular bile salt transport from the basolateral to the canalicular membrane, but the physiological significance of this pathway is not clear (11).

Taken together, the current findings suggest that the features of Na⁺-dependent bile salt uptake into hepatocytes can be largely explained by the molecular properties of Ntcp/NTCP.

B) Sodium-independent uptake via Oatps/OATPs. In contrast to Na⁺-dependent bile salt uptake, Na⁺-independent uptake of bile salts is determined by several different gene products. Oatps/OATPs are multispecific transporter systems with a wide substrate preference for mostly amphipathic organic compounds, including conjugated and unconjugated bile salts, bromosulfophthalein (BSP), bilirubin, DIDS, cardiac glycosides and other neutral steroids, linear and cyclic peptides, mycotoxins, selected organic cations, and numerous drugs such as pravastatin (for review, see Refs. 210, 242, 245). Na⁺-independent bile salt uptake is quantitatively less significant than sodium-dependent uptake and appears to be largely mediated by facilitated exchange with intracellular anions (e.g., GSH, HCO) (210).

In rat liver, basolateral sodium-independent bile salt uptake is mediated by three members of the organic anion transporting protein (Oatp/OATP) family. Other members of the Oatp/OATP family do not appear to be bile salt carrier systems. Oatp1 (Slc21a1) from rat liver was the first cloned member of the Oatp family and is a 670-amino acid glycoprotein with an apparent molecular mass of 80 kDa and 12 putative transmembrane-spanning domains (152). Oatp1 is localized to the basolateral membrane of hepatocytes, the apical membrane of kidney proximal tubular cells, and choroid plexus epithelial cells (16, 28). Developmentally, Oatp1 expression precedes expression of Ntcp, and its mRNA can be detected already at day 16 of gestation in the developing rat liver (91). Oatp1 is a highly versatile and multispecific transport system that transports a wide variety of amphipathic substrates with differing affinities, including bile salts (although with lower affinities than Ntcp), nonbile salt organic anions, organic cations, neutral steroids, and small peptides. More specifically, Oatp1 transports bile salts (taurocholate, cholate, glycocholate, taurochenodeoxycholate, tauroursodeoxycholate, taurodeoxycholate, taurohyodeoxycholate), organic anionic dyes such as BSP, bilirubin monoglucuronide, thyroid hormones including triiodothyronine (T₃) and thyroxine (T₄), steroid hormones (aldosterone, dexamethasone, cortisol) and steroid conjugates [17-estradiol glucuronide, estrone-3-sulfate, dehydroepiandrosteronsulfate (DHEAS)], leukotriene C₄ (LTC₄) and other glutathione conjugates (dinitrophenylglutathione), the anionic magnetic resonance imaging contrast agent gadoxetate (Gd-EOB-DTPA, or gadolinium-ethoxybenzyl-diethylenetriamine-pentacetic acid), neutral steroids such as ouabain, the dipeptidic angiotensin-converting enzyme inhibitors enalapril and temocaprilat, the HMG-CoA reductase inhibitor pravastatin, the peptidomimetic thrombin inhibitor CRC 220, the endothelin receptor antagonist BQ-123, the opioid receptor antagonists D-penicillamine-enkephalin and deltorphin II, the antihistamine fexofenadine, the mycotoxin ochratoxin A, and even organic cations (amphipathic "bulky" type II organic cations) such as N-propylajmalinium and APD-ajmalinium, and to a lesser degree methyl-quinine and recuronium (203, 210, 242, 244, 245). More recently, sulfolithotaurocholate has been identified as a substrate for Oatp1 and Oatp2 (247, 292). Despite this wide substrate specificity, Oatp1 does not mediate uptake of unconjugated bilirubin (210). Antisense studies in oocytes injected with total rat liver mRNA have revealed that Oatp1 can account for 80% of Na⁺-independent taurocholate uptake but for only 50% of BSP uptake of rat liver, consistent with the existence of additional Oatps (126). Oatp1 appears to function as an anion exchanger with HCO (303) and/or GSH (227) as counteranions. Thus continuous GSH efflux from hepatocytes along its in-to-out gradient could provide an efficient driving force for Oatp1-mediated substrate uptake into hepatocytes. Mouse Oatp1 (Slc21a1) has the same substrate specificity as rat Oatp1 and is expressed predominantly in liver with additional expression in kidney (124). Mouse Oatp1 gene has been mapped to chromosome XA3-A5 (124).

Based on homology with Oatp1, a second Oatp2 (Slc21a5) was cloned originally from a rat brain cDNA library. Oatp2 is also highly expressed in liver and kidney (263). Oatp2 is a 661-amino acid protein with an apparent molecular mass of 92 kDa and is 77% identical to Oatp1 (263, 292). Like Oatp1, Oatp2 also has 12 putative transmembrane spanning domains and represents a glycoprotein. Oatp2 is localized to the basolateral membrane of hepatocytes, retina, endothelial cells of the blood-brain barrier, and the basolateral membrane of choroid plexus epithelial cells (105, 292, 331). While Oatp1 is homogeneously distributed throughout the liver lobule as revealed by in situ hybridization and immunofluorescence studies, Oatp2 is predominantly expressed in perivenous/pericentral hepatocytes with sparing of the innermost one to two cell layers (163, 292). Induction of Oatp2 expression by phenobarbital results in a homogeneous distribution throughout the liver (128). Because the major proportion of hepatocellular bile salt uptake normally occurs in the periportal region, Oatp1 is mainly implicated in Na⁺-independent bile salt uptake under normal conditions, while pericentral hepatocytes may become recruited only under cholestatic conditions with downregulation of Ntcp and Oatp1 and spillover of unextracted bile salts form the periportal to the pericentral region (51, 220). The substrate specificity of Oatp2 is similar, but not identical, to that of Oatp1, with similar affinities for the bile salts taurocholate and cholate (203, 210, 242, 245). These include taurocholate, cholate, glycocholate, taurochenodeoxycholate, tauroursodeoxycholate, DHEAS, estradiol 17-glucuronide, T₃ and T₄, ouabain, digoxin, pravastatin, the endothelin antagonist BQ-123, the opioid receptor antagonists D-penicillamine-enkephalin and Leu-enkephalin, biotin, fexofenadine, and the bulky type II organic cations APD-ajmalium and recuronium. Importantly, in contrast to Oatp1, bilirubin monoglucuronide, BSP, LTC₄, and Gd-EOB-DTPA (gadoxetate) are not transported via Oatp2 (203, 210, 242, 245). Moreover, Oatp2 transports ouabain with higher affinity and is unique in mediating high-affinity transport of the cardiac glycoside digoxin (263). Hence, Oatp1 appears to prefer amphipathic organic anions, whereas Oatp2 has an extended substrate preference for neutral compounds (203, 210, 245). Similar to Oatp1, the driving force for Oatp2-mediated uptake appears to be exchange with GSH and GSH conjugates (228).

The third Oatp family member involved in hepatic bile salt uptake in rat liver is Oatp4 (Slc21a10). Oatp4 is the full-length isoform of the rat liver-specific transporter 1 (rLst-1) (58). Although three rLst-1 splice variants have been detected (70), full-length Oatp4 represents quantitatively and functionally the most relevant protein in rat liver (58). Compared with rLst-1, Oatp4 is more highly expressed in liver and contains an additional 35 amino acids, resulting in a predicted 12 transmembrane topology characteristic of all Oatps (58). Oatp4 shares 43% amino acid identity with Oatp1 and 44% identity with Oatp2 (210, 245). While rLst-1 transports only taurocholate, Oatp4 also transports BSP, estrone-3-sulfate, estradiol 17-glucuronide, DHEAS, prostaglandin E₂, LTC₄, the thyroid hormones T₃ and T₄, and gadoxetate (58). Similar to Oatp1 and Oatp2, Oatp4 is a multispecific transporter with high affinities for BSP, DHEAS, LTC₄, and anionic peptides. In addition, Oatp4 appears to be particularly involved in the hepatic clearance of anionic peptides including microcystin (a toxin derived from algae) and cholecystokinin (a gastrointestinal peptide hormone) (210, 245).

Oatp3 (Slc21a7) is not expressed in liver in contrast to initial reports (1) but may be important for intestinal bile salt uptake (370) (see sect. ID). Oatp3 has a similar broad substrate specificity but much lower affinities than Oatp4 (59). Thus, while Oatp4 works in concert with Oatp1 and Oatp2 in the basolateral membrane of rat hepatocytes, Oatp3 is a multispecific transport system in the small intestine (59).

An evolutionary ancient liver specific Oatp has been characterized in a primitive vertebrate, the small skate (Raja erinacea) with substrate similarities to rat Oatp4 and human OATP-C (55). This Oatp shares only ~40-50% amino acid identity with other liver-specific OATPs/Oatps and is most closely related to human OATP-F in brain. These findings emphasize the early evolutionary development of the Slc21a/SLC21A transporter family.

Taken together, the transport characteristics of Oatp1, -2, and -4 can account for the bulk of Na⁺-independent bile salt uptake in liver (210, 245).

In humans, three liver-specific OATPs (OATP-A, OATP-C, and OATP-8) transport bile salts (208). For bile salt uptake, OATP-C is the most relevant isoform (210, 245).

The first human OATP identified in human liver was OATP-A (SLC21A3) (207). It consists of 670 amino acids and exhibits only a 67% amino acid homology to rat Oatp1 (which is not an ortholog as suggested by low amino acid identity and different substrate specificity), 73% with Oatp2, 42% with Oatp4, and 44% with human OATP-C. Although OATP-A was originally cloned from human liver (207), its hepatic expression level is low, and its contribution to overall hepatic bile salt uptake is probably minor (245). It is predominantly expressed in human cerebral endothelial cells where it may play a role in the blood-brain barrier (104). OATP-A transports bile salts such as taurocholate, cholate, tauroursodeoxycholate, in addition to BSP, estrone-3-sulfate, DHEAS, the magnetic resonance imaging contrast agent Gd-B 20790, the opioid receptor antagonists D-penicillamine-enkephalin, and deltorphin II, fexofenadine, and the bulky type II organic cations APD-ajmalium, recuronium, N-methylquinine, and N-methylquinidine. Transport rates were highest for the organic cation N-propylajmalium and the peptidomimetic drug CRC 220. In addition, OATP-A mediates unique high-affinity transport of the bulky lipophilic organic cations methylquinine and methylquinidine (210, 245).

OATP-C (SLC21A6) also known as LST-1 and OATP2 consists of 691 amino acids and is selectively expressed at the basolateral membrane of hepatocytes (2, 142, 189). OATP-C exhibits the highest amino acid identity (64%) with rat Oatp4 (210). As a result of gene duplication in humans, both OATP-C and OATP-8 represent the human orthologs of rodent Oatp4. OATP-C transports taurocholate (with slightly lower affinity than NTCP), bilirubin monoglucuronide, conjugated steroids, eicasonoids, thyroid hormones, and peptides (210). The substrate specificity of OATP-C is very comparable to rat Oatp4 and, more specifically, includes taurocholate, bilirubin monoglucuronide, BSP, estrone-3-sulfate, estradiol 17-glucuronide, DHEAS, prostaglandin E₂, thromboxane B₂, LTC₄ and LTE₄, the thyroid hormones T₃ and T₄, and pravastatin (210). Although OATP-C exhibits large overlapping substrate specificities with other OATPs of human liver (208), a unique property is its capacity to transport unconjugated bilirubin (77, 78). In contrast to OATP-A, OATP-C shows a substrate preference for organic anions and does not include amphipathic organic cations. Of note, OATP-C transports taurocholate with slightly lower affinity than NTCP, although current data suggest that it represents an important Na⁺-independent bile salt uptake system in human liver (208, 210). Polymorphisms in OATP-C associated with variable degrees in plasma membrane expression may represent an important factor influencing drug disposition (354). In addition, a recently identified OATP-C mutation severely interferes with normal basolateral OATP-C expression and function (J. König and D. Keppler, personal communication).

OATP8 (SLC21A8) is 80% identical with OATP-C and is also expressed at the basolateral membrane of human hepatocytes (190). Thus OATP-C and -8 are expressed in a liver-specific fashion. The data on bile salt transport by OATP8 are controversial since bile salts were not transported when OATP8 was expressed in mammalian cells (190), but they were when OATP8 was expressed in oocytes (208, 210). Interestingly, OATP8 (and rat Oatp4) has been identified as hepatic uptake systems for cholecystokinin (148).

OATP-B (SLC 21A9) is also expressed at the basolateral membrane of human hepatocytes but does not transport bile salts (208, 351). Although OATP-B is predominantly expressed in liver, it is not liver specific and is found in many other tissues (208) including placenta (341) (see sect. IF).

Hence, although sodium-independent bile salt transport is an intrinsic feature of several Oatps/OATPs, each of these polyspecific transport systems exhibits additional substrate specificities and preferences that may even be specific for certain substrates (e.g., digoxin for Oatp2 and OATP8). For many of these substrates, Oatp/OATP-mediated uptake appears to be the main if not the only uptake route, based on the similarities of K_m values between oocyte expression systems and total liver.

Basolateral efflux systems belonging to the multidrug resistance (MRP/Mrp) subfamily normally are expressed at only very low levels but may be induced under cholestatic conditions. In humans, this family currently comprises six members (MRP1-6), at least four of which (MRP1, -2, -3, -6) have been demonstrated in liver (37, 180). The hepatic expression of MRP4 and MRP5 proteins is low, and their physiological function and (sub)cellular localization are not yet known (140). Three members of this family have also been identified in rat (Mrp2, -3, -6), five in mouse (Mrp1, -2, -4, -5, -6), and one in rabbit (Mrp2) (180). The founding member of the MRP family, MRP1 (Mrp1 in rodents), was initially cloned from a multidrug resistant small lung cancer cell line (73) and is minimally (if at all) expressed in normal liver (73), where it has been reported to be localized to the basolateral membrane of hepatocytes (240). However, potential antibody cross-reactivity with other Mrps should be considered when interpreting these early reports. Mrp3/MRP3 and Mrp6/MRP6 have also been localized to the basolateral membrane of hepatocytes, while Mrp2/MRP2 is localized to the canalicular membrane. At the hepatocellular level, rat Mrp3 is normally only expressed in the terminal cells in the lobule surrounding the pericentral vein (89, 192, 331). Mrp3/MRP3 has also been localized to the basolateral membrane of cholangiocytes (89, 194, 299, 308, 331) and intestinal epithelia (135, 186, 300, 308), where it may be involved in the cholehepatic and enterohepatic circulation of bile salts. Under normal conditions, expression of Mrp3/MRP3 is also very low in hepatocytes in contrast to cholangiocytes. Human MRP3 is mainly expressed in the periportal region of the lobule in addition to cholangiocytes (37, 308). Similar to Mrp2/MRP2, Mrp1/MRP1 and Mrp3/MRP3 are ATP-dependent export pumps whose spectrum (although with different affinities) include glucuronide and glutathione conjugates of endogenous and exogenous compounds (180, 191). Mrp1/MRP1 (179, 180) and Mrp3/MRP3 (6, 137, 138) have been shown to transport divalent bile salts such as sulfated taurolithocholate and taurochenodeoxycholate with high affinity. In addition, Mrp3 can also transport monovalent bile salts such as tauro- and glycocholate (6, 137, 138), while human MRP3 transport only glycocholate with low affinity, but not taurocholate to a significant degree (5, 398). Mrp1 and Mrp3/MRP3 are normally expressed at very low levels in hepatocytes, but are dramatically upregulated in cholestasis in rats (89, 136, 268, 285, 331, 352, 367). Because Mrp1/MRP1 and Mrp3/MRP3 are able to transport the divalent anionic bile salts such as sulfated and glucuronidated bile salts that are eliminated into urine in cholestasis, the induction of Mrp1 and Mrp3/MRP3 during cholestasis may explain the shift toward renal excretion of bile salts as a major mechanism for bile salt elimination in patients with chronic, long-standing cholestasis (290, 339). Similarly, Mrp4 RNA has been recently shown to be upregulated in bile salt-fed mice, suggesting that this transporter could also be involved in basolateral bile salt efflux (314). However, further studies will have to demonstrate whether Mrp4 is localized to the basolateral membrane of hepatocytes. Mrp6 is localized predominantly to the lateral membrane of hepatocytes in rat liver where it mediates transport of the anionic cyclopentapeptide and endothelin receptor antagonist BQ-123; of note, other cyclic peptides such as endothelin and vasopressin are transported by Mrp2 but not by Mrp6 (233). MRP6 is constitutively and highly expressed in hepatocytes and kidney (193). Recent evidence suggests that MRP6 may be involved in transport of glutathione conjugates and leukotrienes (LTC₄) (146). Mutations of the MRP6 gene are associated with pseudoxanthoma elasticum, although the functional link underlying this observation remains unclear (27, 146, 294).

Members of the Oatp/OATP family also remain candidates for bile salt efflux at the basolateral membrane, since studies in Xenopus laevis oocytes have shown that Oatp1 and -2 are able to operate as bidirectional exchangers (228), although this remains to be demonstrated for hepatocytes.

The mechanisms involved in the transcellular movement of bile salts across hepatocytes from the basolateral to the canalicular membrane are poorly understood. With physiological bile salt loads, a considerable fraction is bound to intracellular binding proteins (e.g., glutathione-S-transferases, 3-hydroxysteroid dehydrogenase, fatty acid-binding proteins in rat liver; a 36-kDa bile acid binding protein in human liver) and diffuses to the canalicular membrane in a protein bound form (for review, see Ref. 4). In addition, free, unbound intracellular bile salts may reach the canalicular membrane by rapid diffusion. At high bile salt loads, increasing partition of hydrophobic bile salts occurs into intracellular organelles including endoplasmatic reticulum, Golgi apparatus, and other membrane-bound compartments (4). Early reports of a direct vesicle-dependent fraction of transcellular bile salt transport can now be better explained by vesicular targeting of their respective transport systems (183).

Canalicular bile secretion represents the rate-limiting step in bile formation. The canalicular membrane contains both ATP-dependent and ATP-independent transport systems (Table 1, Fig. 1). ATP-dependent transport systems in the liver are members of the ATP-binding cassette (ABC) superfamily, and they transport biliary constituents against steep concentration gradients across the canalicular membrane that are often in the range of 100- to 1,000-fold and are driven by ATP hydrolysis. A typical ABC transporter consists of 12 or more membrane-spanning domains that determine the substrate specificity and two intracellular nucleotide-binding loops that are highly conserved and contain the Walker A and B motifs for binding and hydrolysis of ATP (140). Most canalicular ABC proteins belong to the multidrug resistance P-glycoprotein (MDR) family also known as ABC-B family or the multidrug-resistance protein (MRP) family (ABC-C) (140, 178). Two other families, ABC-A and ABC-G, may also be relevant, since at least three ABC-A members (ABCA1, -2, -3) and three ABC-G half transporters (ABCG2, -5, and -8) are highly expressed in liver (94, 229).

The canalicular membrane contains a bile salt export pump (Bsep/BSEP) for monovalent bile salts, a conjugate export pump (Mrp2/MRP2) for divalent bile salts, and various other amphipathic conjugates, including GSH (a major determinant of bile salt-independent canalicular bile flow), a multidrug export pump (Mdr1/MDR1) for bulky amphipathic organic cations (e.g., various drugs), a phospholipid flippase (Mdr2 in rodents/MDR3 in humans) for phosphatidylcholine, a P-type ATPase (Fic1/FIC1) mutated in hereditary cholestasis but whose function is still unknown, and a Cl/HCO exchanger (AE2) for HCO excretion. The latter represents the only functionally relevant ATP-independent transport system at the canalicular membrane (238, 243). All other transporters, except FIC1 (which is a P-type ATPase), belong to the ABC superfamily. AE2 and MRP2 are the major driving forces for bile salt-independent bile flow, while BSEP drives bile salt-dependent flow (205, 258, 259, 342, 359).

A) Canalicular excretion of monovalent bile salts via Bsep/BSEP. The canalicular bile salt export pump Bsep/BSEP (Abcb11/ABCB11) originally was known as the sister of P-glycoprotein (Spgp/SPGP). Following the partial cloning of Spgp in pig liver (68), the full-length cDNA was cloned from rat liver (69, 112). Functional expression of rat Spgp in Sf9 insect cells demonstrated that Spgp functions as an ATP-dependent bile salt transporter with transport characteristics comparable with canalicular rat liver plasma membrane vesicles (112). These functional data, together with hepatocyte-specific expression of Spgp on the surface of canalicular microvilli, indicate that Spgp represents the mammalian canalicular bile salt export pump. The rat Bsep is a 1,321-amino acid protein with 12 putative membrane-spanning domains, four potential N-linked glycosylation sites, and a molecular mass of ~160 kDa. Bsep was localized by immunofluorescence microscopy and immunogold labeling studies to canalicular microvilli and to a canalicular subpopulation of membrane vesicles (112). When ontogenic expression of Bsep was compared with that of Mrp2, it became evident that Bsep is almost undetectable before birth, whereas Mrp2 was already observed in livers of 16-day-old fetuses (400), indicating that biliary excretion of monovalent bile salts does not take place until birth. Bsep transports various bile salts at rates in the same order of magnitude as ATP-dependent transport in canalicular rat liver plasma membrane vesicles. In addition to taurocholate, Bsep also mediates ATP-dependent transport of glycocholate, taurochenodeoxycholate, and tauroursodeoxycholate (210, 245). Rat Bsep mediates low-level resistance to taxol, but not to other drugs (e.g., vinblastine, digoxin) that form part of the multidrug resistance phenotype (69). Transfection studies with mouse Bsep also revealed only slightly enhanced vinblastine efflux, indicating that Bsep is probably not of major relevance for detoxification of xenobiotics (218).

The mouse Bsep gene was localized to a region on mouse chromosome 2, which has also been linked to genetic gallstone susceptibility, although the detailed mechanisms remain unclear (117, 213). In contrast to humans with a hereditary BSEP defect (PFIC-2), targeted inactivation of this gene in mice results in nonprogressive but persistent intrahepatic cholestasis, due to the de novo formation and biliary excretion of muricholic acid and a novel tetra-hydroxylated bile salt in mice (376). Of note, bile flow is minimally reduced and bile salt excretion is only reduced to 30%, suggesting that additional bile salt transporter(s) may exist in mice. Although the molecular identity of this alternative transport system remains to be determined, Mrp2 is a likely transporter for tetrahydroxy bile salts (245). Alternatively, it remains possible that sulfated and glucuronidated dianionic bile salts, which also are Mrp substrates, account for most of the secreted bile salts in these animals (180). Mouse Bsep contains several phosphorylation sites that appear to be involved in regulating bile salt transport capacity by Bsep (264).

Bsep is highly conserved during vertebrate evolution as suggested by the recent cloning of a Bsep ortholog from the liver of the small skate, Raja erinacea, a 200 million-year-old marine vertebrate (23, 54). Notably, the sites of published BSEP mutations in humans are also conserved from the skate Bsep ortholog, and mutations of several of these sites inhibit skate Bsep transport function in Sf9 cells (54).

Identification of the gene responsible for a subtype of progressive familial intrahepatic cholestasis (PFIC-2) has led to the discovery of the human BSEP gene. The human BSEP gene is mutated in patients with PFIC-2, characterized by absence of BSEP from the canalicular membrane and extremely low biliary bile salt concentrations that are <1% of normal (see sect. IIIA) (155, 344). This suggests that BSEP is the major canalicular bile salt transport system. After several years of effort, the human BSEP has now been functionally expressed by two groups and appears similar to studies in human canalicular membrane vesicles with respect to bile salt affinity and substrate specificity (53, 265).

B) Canalicular excretion of divalent bile salts via Mrp2/MRP2. The bilirubin conjugate export pump (Mrp2/MRP2) (Abcc2/ABCC2), functionally also known as the canalicular multispecific organic anion transporter (cMOAT), was originally cloned from rat liver (49, 280), followed by human (353) and mouse liver (101). Mrp2/MRP2 mediates the canalicular excretion of a broad range of organic anions; most of these are divalent amphipathic conjugates with glutathione, glucuronate, and sulfate formed by phase II conjugation in the hepatocyte (e.g., bilirubin diglucuronide) (76, 191, 274). Canalicular excretion of GSH is also mediated through Mrp2/MRP2 (279). Divalent bile salts with two negative charges such as sulfated tauro- or glycolithocholate are transported by Mrp2/MRP2, whereas monovalent bile salts are not substrates for Mrp2/MRP2 (179, 180). However, mutation of a single amino acid confers transport capacity for monovalent bile salts to Mrp2 (149). Other Mrp/MRP isoforms such as Mrp1/MRP1 and Mrp3/MRP3 are located at the basolateral membrane where they may serve as a compensatory overflow system under cholestatic conditions when canalicular (Mrp2/MRP2) excretory function is impaired. The mouse Mrp2 gene was localized to a region on mouse chromosome 19D2 (214, 363), which has also been linked to genetic gallstone susceptibility (213). The human MRP2 gene has been mapped to chromosome 10q24 (363). Mutations of the MRP2 gene result in the Dubin-Johnson syndrome characterized by impaired canalicular excretion of a broad range of endogenous and exogenous amphipathic compounds (see sect. IIIA); the rat model for this syndrome is the transport deficient (TR) and Groningen Yellow (GY) rat (Wistar strain) or Eisai hyperbilirubinemic (EHBR) rat (Sprague-Dawley strain), which were pivotal models for the functional characterization of cMOAT and subsequent cloning of Mrp2 (281).

C) Other canalicular transport systems involved in bile secretion and their relation to bile salt excretion. Once bile salts have been excreted into bile, they stimulate the release of phosphatidylcholine and cholesterol from the outer leaflet of the canalicular membrane (272), which then in turn form mixed micelles in bile. By doing so, bile salt toxicity to the bile duct epithelium is avoided, which would otherwise occur due to an unopposed detergent bile salt action. The phospholipid flippase Mdr2/MDR3 (Abcb4/ABCB4) ensures the continuous supply of phosphatidylcholine to the outer leaflet of the canalicular membrane (327, 362).

In addition to biliary excretion of phospholipids, bile is also a major pathway for elimination of cholesterol, which is mostly derived from plasma high-density lipoprotein (HDL). Hepatocellular uptake of HDL cholesterol is mediated via a scavenger receptor (SR-BI) (3, 158, 195). An ATP-dependent cholesterol transporter (ABCA1) mediates reverse cholesterol transport from macrophages into HDL particles, removes absorbed cholesterol from enterocytes, and is mutated in individuals with Tangier disease and familial HDL deficiency with an increased risk for atherosclerosis and premature coronary artery disease (34, 48, 301). ABCA1 is highly expressed in liver but probably does not play a major role in biliary cholesterol excretion, since Abca1 knock-out mice also do not demonstrate defects in bile salt-induced cholesterol excretion (A. Groen, personal communication). Mutations of two highly homologous genes (ABCG5 and ABCG8) that encode the plant sterol sitosterol transporters, sterolin-1 and -2, respectively, have been identified in patients with sitosterolemia, a disease characterized by impaired biliary excretion of dietary sterols. ABCG5 and ABCG8 are also highly expressed in liver (26, 144, 222, 229) and may play a key role in hepatic cholesterol excretion (222, 273) as suggested by a fivefold increased biliary cholesterol excretion in mice with transgenic over expression of human ABCG5 and ABCG8 (397).

Fic1/FIC1 (ATP8B1) a P-type ATPase mutated in "familial intrahepatic cholestasis" belongs to a family of putative aminophospholipid transporters. Fic1/FIC1 has been localized to the canalicular membrane and the bile duct epithelium (361) but is also highly expressed in extrahepatic tissues including the intestine and pancreas (50, 96). The detailed function of Fic/FIC1 is unclear, but mutations of this transporter result in variants of familial intrahepatic cholestasis, suggesting that it must play an important direct or indirect role in canalicular bile salt excretion (50). Of note, these patients have a prominent reduction in the biliary excretion of hydrophobic bile salts such as lithocholate and chenodeoxycholate relative to cholate conjugates, indicating that Fic1/FIC1 could directly excrete highly hydrophobic bile salts (335). Apart from this, Fic1/FIC1 could also play an indirect role in bile secretion by maintaining the canalicular membrane asymmetry between the inner and the outer layer by maintaining phosphatidylethanolamine and serine within the inner bilayer of the plasma membrane or regulating the docking of vesicles fusing with the canalicular membrane (276). Strong expression of Fic1/FIC1 in extrahepatic tissues such as pancreas, small intestine, and kidney suggests a more general role in the regulation of secretory processes (50) and may explain some of the extrahepatic features associated with these syndromes such as pancreatitis, diarrhea, and nephrolithiasis. Fic1 (/) mice have normal biliary bile salt excretion but accumulate bile salts when fed orally due to abnormal regulation of intestinal bile salt absorption. The detailed mechanisms remain to be resolved (283).

Although contributing only 3-5% to the total liver cell mass, bile duct epithelial cells (cholangiocytes) play an important role in normal bile secretion (39, 355). Large and medium-sized but not small (<30 µm in diameter) intrahepatic bile ducts contain several transport systems for secretory and absorptive functions (168) (Table 1, Fig. 1). Biliary constituents including bile salts, glucose, and drugs may be transported from bile into cholangiocytes However, only a minority of bile salts are in solution in bile as free monomers at the level of the bile ducts, and thus only small amounts of bile salts might be available for absorption by cholangiocytes (245).

Bile salts are taken up by Isbt/ISBT, also called Asbt/ASBT (SLC10A2) (216), which is identical to the gene product expressed in the terminal ileum. However, the relative abundance of Isbt in rat cholangiocytes is about sevenfold lower than in the ileum (8, 216), possibly indicating different physiological roles such as signaling through bile salts. A minor part of reabsorbed bile salts may also be taken up in their protonated form by nonionic diffusion (139).

The gallbladder epithelium has considerable capacity to modify the composition of primary hepatic bile by absorptive and secretory processes. MRP2 and MRP3 were recently identified in human gallbladder epithelia. MRP2 was located in the apical membrane and MRP3 in the basolateral membrane as in other epithelia. Of note, MRP1 protein expression was not detectable (299). Moreover, ISBT and OATP-A expression were detected in a human gallbladder-derived biliary epithelial cells where these transporters may mediate sodium-dependent and -independent taurocholate uptake (67).

After uptake, bile salts are effluxed via the basolateral membrane of cholangiocytes into the peribiliary plexus. Functionally, this mechanism has been characterized as Na⁺ independent (25). At a molecular level, this may be achieved by Mrp3/MR3, which has been identified at the basolateral membrane of cholangiocytes and gallbladder epithelium in rats and humans (89, 194, 299, 308, 331).

Additional transport systems may involve an alternative (truncated) splicing variant of Isbt/Asbt (t-Asbt) for which no driving force has as yet been established (217). Expression studies in oocytes predict that t-Asbt in contrast to Asbt acts as an efflux carrier (217). The latter observation would suggest that two Asbt isoforms are required for cholangiocellular bile salt transport to occur, although the existence of t-Asbt is being questioned recently. Alternatively, Oatp3 could be involved in basolateral bile salt efflux from cholangiocytes (210).

Bile salts, cholesterol, and phospholipids undergo extensive enterohepatic cycling/enterohepatic circulation, thereby returning these biliary constituents to the liver for reexcretion into bile (139, 156, 245) (Table 1, Fig. 1). The most efficient bile salt conservation mechanism is the uptake of conjugated bile salts in the terminal ileum via a Na⁺-dependent mechanism (74, 342). In addition, a Na⁺-independent anion exchanger has been identified in proximal rat jejunum (12). Passive diffusion of unconjugated bile salts also occurs in small and large intestine (246).

Na⁺-dependent bile salt uptake occurs via the ileal bile salt transporter (Isbt/ISBT or Ibat/IBAT) also known as the apical sodium-dependent bile salt transporter (Asbt/ASBT). This transport system has also been identified in the apical membrane of cholangiocytes and proximal renal tubular cells. Isbt was originally cloned from hamster intestine (387), followed by human (388), rat (320), rabbit (198), and mouse (302) ileum. Size (48 kDa), membrane topology, and transport characteristics are similar to Ntcp/NTCP (35% amino acid identity) (79, 129, 342). Isbt is expressed biphasically during rat development, with the first expression on day 22 of gestation, followed by a transient decrease and a sharp increase at postnatal days 17 and 18 (320, 322). Both primary and secondary conjugated and unconjugated bile salts are substrates for Isbt/ISBT, the highest affinity being reported for conjugated dihydroxy bile salts (267). However, in contrast to Ntcp/NTCP, which transports some nonbile salt substrate in addition to bile salts, the substrate specificity of Isbt/ISBT appears to be strictly limited to bile salts. ISBT is the major intestinal bile salt uptake system in humans as emphasized by ISBT mutations that result in bile salt malabsorption (267, 388).

The Na⁺-independent bile salt transporter Oatp3 (Slc21a7) is 80-82% identical to Oatp1 and Oatp2 and has transport characteristics similar to Oatp2, with a range of amphipathic anions as its substrates, including bile salts (59, 370). Oatp3 was originally cloned from retina and was found to be expressed in the brush-border membrane of jejunal enterocytes (1, 370). Oatp3 mRNA transcripts were detected throughout the entire small intestine (as well as brain, lung, and kidney), but Oatp3 protein is predominantly located to the apical surface of jejunal epithelial cells (1, 370), consistent with a role of Oatp3 as the Na⁺-independent transport system which has been functionally localized to the jejunum. Bile salt uptake into jejunal brush-border membrane vesicles is stimulated by an in-to-out HCO gradient, similar to Oatp1 (303). However, it remains to be determined whether this mechanism also applies to Oatp3. The relative importance of Oatp3 for intestinal bile salt uptake compared with Isbt also remains to be clarified (342). Interestingly, OATP-A has been proposed as the human Oatp3 ortholog (370), and human OATP-A has been detected in human intestine (245).

In addition to uptake systems in the apical membrane, the apical brush-border membrane also contains excretory systems, including members of the Mdr and Mrp family (e.g., Mdr1, Mrp2) (Fig. 1). As far as bile salt transport is concerned, Mrp2 could play a role as an alternative excretory route for sulfated/glucuronidated bile salts (in analogy to the role of Mrp2 in kidney, see sect. IE).

As with other cells involved in bile salt transport, the information about the molecular mechanisms of intracellular bile salt transport in enterocytes is limited. Photoaffinity labeling studies have identified a 14-kDa cytosolic intestinal bile acid-binding protein (I-Babp) that is cytoplasmatically attached to Isbt (115, 196). I-Babp probably represents the most important protein for transcellular bile salt movement through enterocytes (197). The functional ileal bile salt uptake complex is multimeric and comprises four Isbt dimers and four I-Babps (196). Similar ontogenic expression patterns of Isbt and I-Babp as well as their response to bile salts and dexamethasone suggest that both transport systems might be controlled by similar if not identical regulatory mechanisms (145, 320).

At a functional level, an anion exchange mechanism has been demonstrated at the basolateral membrane of intestinal cells (381). Recently t-Asbt (which can function as an anion exchanger) has been reported to be expressed twofold higher at the mRNA level than the full-length Asbt in rat ileum (217). Another potential candidate for bile salt efflux from enterocytes is Mrp3/MRP3, which has also been identified in both rat and human small intestine (135, 186, 300, 308). Mrp3/MRP3 is expressed in the basolateral membrane in all intestinal segments but is lowest in duodenum and markedly increased in the terminal ileum and colon (330). This is in contrast to Mrp2/MRP2, which is expressed primarily in the apical membrane of the proximal intestine (65, 300, 348). The high expression of Mrp3/MRP3 in terminal ileum provides a mechanism to return bile salts to the portal circulation. Mrp3/MRP3 may also play an important role in drug absorption in the intestine.

Bile salts that escape first-pass clearance by the liver are filtered at the glomerulus from plasma into urine, where they are reabsorbed in the proximal convoluted tubule (386) (Table 1, Fig. 1). Thus, under normal conditions, urinary bile salt losses are minimized. Under cholestatic conditions however, renal excretion of bile salts may become a major alternative elimination route for elimination of (mainly divalent sulfated and glucuronidated) bile salts (290), which may be attributed to increased passive glomerular filtration of (elevated) serum bile salts and active tubular excretion of (mainly divalent) bile salts, together with reduced tubular bile salt conservation.

The apical plasma membrane of the proximal renal tubular cells contains transport systems for bile salt reabsorption as well as excretion into urine (342). Bile salts are reabsorbed by the apical Na⁺-dependent bile salt transporter Isbt in the proximal convoluted tubule (71). In addition to Isbt, Mrp2 (306) and Oatp1 (28) have also been localized to the apical brush-border membrane. Mrp2 may be involved in tubular excretion of organic anions (e.g., para-aminohippuric acid) under normal conditions and increased urinary excretion of sulfated/glucuronidated bile salts under cholestatic conditions (306). In contrast to its basolateral localization in hepatocytes, Oatp1 is localized to the apical membrane of the S3 segment of proximal renal tubules (28). The relative contribution of Oatp1 to tubular bile salt uptake remains unclear. Moreover, Oatp3 (Slc21a7) has also been reported to be expressed in rat kidney (1), although this remains controversial (342). By analogy to the rat, ISBT (74), OATP-A (207), and MRP2 (305) have also been localized in human proximal kidney tubular cells.

Little is known about the basolateral counterparts of the apical transport systems described above. Recent evidence suggests a potential role for Mrp1 (285). However, Mrp3 has recently been localized to the basolateral membrane of the proximal renal tubule in rats (330). Therefore, it will also be of particular interest to see whether Mrp3 also plays an important role for basolateral efflux, e.g., of bile salts.

Because the fetal liver is immature and ontogenetic expression of hepatobiliary transporters is not detectable until shortly before birth (19), bile salts undergo minimal biliary excretion by the fetal liver, although bile salts are synthesized by the fetal liver in utero (260). Instead, they are eliminated by the maternal liver after vectorial translocation from the fetal to the maternal circulation via the placenta ("placenta-maternal liver tandem"). The blood-placental barrier of term placenta begins at the fetal side with the endothelial cells of fetal capillaries, cytotrophoblast (absent in term placenta), and syncythiotrophoblasts that face the maternal blood with their apical surface. Distinct transport systems have been identified at a functional level at the basolateral (fetal-facing) and apical (maternal-facing) membrane of the trophoblast. However, the molecular identity of these transport systems has not yet been resolved, and data are still quite fragmentary (342).

Bile salt uptake occurs via Na⁺-independent, bidirectional, and HCO trans-stimulatable mechanisms, consistent with a role for OATPs/Oatps. Notably, partial OATP-A (SLC21A3) transcripts have been identified in term placenta (342). Recently, OATP-B (SLC21A9) was localized to the basal syncythiotrophoblast and cytotrophoblast membranes, where it may be involved in the placental uptake of fetal-derived sulfated steroids (341). Preliminary data indicate the presence of Oatps 1, 2, and 4 mRNA transcripts in rat placenta (J. J. Marin, personal communication). Moreover, recent studies have localized MRP3 in fetal blood endothelia of term placenta and syncytiotrophoblast layer, suggesting a potential role for MRP3 in the extrusion of fetal bile salts through endothelial and syncythiotrophoblast barriers (343). Similarly Mrp1, -2, and -3 transcripts have recently been detected in rat placenta, although their subcellular localization remains to be determined (Marin, personal communication).

Both ATP-dependent and ATP-independent bile salt transport systems have been identified, the latter appearing to be the predominant transport mechanism (46, 342). However, BSEP remains a candidate, since partial Bsep/BSEP transcripts have been identified in term placenta (342).

	II. REGULATION OF BILE SALT TRANSPORTERS IN NORMAL PHYSIOLOGY

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The functional expression of membrane transport proteins can be regulated at several levels, including gene transcription, events that control translation of RNA, and posttranscriptional activity (see Table 2 and Figs. 2 and 3). Although the mechanisms that control gene transcription of membrane transporters are still incompletely understood, a number of factors may be involved including constitutive and inducible DNA binding proteins that bind to upstream regulatory elements (RE) in the gene promoters. These transcription factors often act together to either stimulate or inhibit gene expression (17). Recent interest has focused on a group of nuclear hormone receptors (NHR) that are activated by various ligands such as steroids, retinoids, and hormones (61, 62). The nuclear receptor superfamily is divided into four major subgroups based on their dimerization and DNA binding properties (for reviews, see Refs. 66, 169, 236, 237). Class I contains the classical steroid hormone receptors such as for estradiol, progesterone, testosterone, cortisol, and aldosterone. Class II receptors comprise the receptors for vitamin D₃ (which recently has been shown to be also activated by the hydrophobic bile salt lithocholate, Ref. 234), T₃, and all-trans-retinoic acid. The latter (all-trans-retinoic acid receptor RAR) is also involved in the regulation of hepatobiliary transporter genes as outlined below. Other class II receptors involved in the regulation of bile salt synthesis and transport include the farnesoid X receptor (FXR) that responds to bile salts; peroxisome proliferator-activated receptor (PPAR), whose high-affinity ligands are fatty acids, eicosanoids, fibrates, and NSAIDS; liver X receptor (LXR) that binds oxysterols; and the pregnane X receptor (PXR) whose human ortholog is the steroid and xenobiotic receptor (SXR) (392). PXR and SXR receptors are the targets for catatoxic steroids and certain xenobiotics. Class II receptors heterodimerize with the retinoid X receptor (RXR) enabling high-affinity DNA binding to direct repeats (DR) separated by spacer nucleotides of variable number (DR1-5), which then leads to activation of gene transcription. Because heterodimer formation largely depends on the availability of RXR, limiting cellular/nuclear RXR concentrations may result in a trans-repressive effect.

In addition to these ligand-activated nuclear receptors, other factors such as the hepatocyte nuclear factor (HNF) family of liver-enriched transcription factors including HNF-1 (318, 319), HNF-3 and CCAAT/enhancer binding protein (C/EBP), as well as sterol responsive element binding protein (SREBP) and nuclear factor kappa B (NF-B) also appear to play an important role in the regulation of hepatobiliary transporter expression (60, 257, 360) (Table 2).

A.  Transcriptional Regulation of Bile Salt Transporters

1.  Role of nuclear hormone receptors in regulation of bile salt transporters

Table 2 and Figure 2 list several of the bile salt transporters whose expression appears to be regulated by one or more NHRs. Bile acids are ligands for the nuclear hormone receptor FXR, which together with its heterodimeric partner, the RXR, acts as a transcription factor for several bile salt transporters, including the hepatic bile salt export pump Bsep and the ileal bile acid binding protein I-Babp (235, 277, 373). In addition, the expression of short heterodimeric protein (SHP)-1, which acts as a transcriptional repressor is itself regulated by FXR and can downregulate the expression of several genes including Ntcp and cholesterol-7a-hydroxylase CYP7A1, the rate limiting enzyme in bile salt synthesis. Recent studies suggest that the bile salt lithocholate and its 6-hydroxylated metabolite are also ligands for the PXR, resulting in the transcription of CYP3A, an enzyme involved in hydroxylation and detoxification of bile salts (334, 393). Two ₁-fetoprotein transcription factor (FTF) also known as liver receptor homolog-1 (LRH-1)-like elements have also been described in the promoter region of MRP3 that function as bile salt response elements (147). Thus a picture is emerging where bile salts may regulate the expression of several important bile salt-transporting and -metabolizing systems by binding to various nuclear receptors (Fig. 2).

View larger version (20K): [in this window] [in a new window]   Fig. 2. Role of ligand-activated transcription factors in the transcriptional regulation of bile salt synthesis and transport. Conversion of cholesterol to bile salts by CYP7A1 and 8B1 is stimulated by oxysterol-activated liver X receptor (LXR). Bile salts inhibit their own synthesis by farnesoid X receptor (FXR)-dependent activation of SHP-1, which in turn suppresses CYP7A1 and 8B1 transcription. Bile salts also suppress retinoic acid receptor (RAR)-dependent genes such as Ntcp via the same pathway. Activation of FXR by bile salts stimulates transcription of Bsep, Mrp2, I-Babp, and OATP8. Activation of pregnane X receptor (PXR) by bile salts induces transription of Oatp2 and Mrp2. Finally, liver receptor homolog-1 (LRH-1) may mediate induction of MRP3 by bile salts in human enterocytes. RXR, retinoid X receptor; FTF, fetoprotein transcription factor.

A) RXR. RXR is the obligate heterodimeric partner for the class II nuclear receptors and is activated by binding of 9-cis-retinoic acid. Thus alterations in the expression of RXR would be capable of influencing the expression of a large number of bile salt synthesis and transporter genes. RXR is highly expressed in liver, kidney, muscle, lung, and spleen, whereas two other RXR isoforms, RXR- and -, are not as highly expressed (371).

Although RXR- knock-out mice do not survive beyond midgestation, the RXR- gene has been deleted in adult animals specifically in the liver using cre-mediated recombination under the influence of an albumin promoter (371). These studies demonstrate that multiple metabolic pathways in the liver that are regulated by RXR heterodimerization are markedly altered when RXR- is absent. These include FXR, which functions with RXR to enhance SHP expression, thereby suppressing the conversion of cholesterol to bile salts. The absence of RXR led to a significant increase in CYP7A1 mRNA (371). RXR/FXR appears to have a dominant negative influence on CYP7A1 expression, since RXR/LXR normally promotes the expression of CYP7A1 to stimulate conversion of cholesterol to bile salts. Other class II nuclear receptors that influence hepatic metabolic pathways are also impaired in the absence of RXR- including RXR/CAR- and RXR/PXR (371), leading to marked reductions in mRNA levels for liver fatty acid binding proteins and CYP4A1 and CYP2B10 and CYP3A1.

Thus the expression of RXR is one mechanism through which a coordinated effect on multiple gene products that influence bile salts, fatty acid, cholesterol, steroid, and xenobiotic metabolism, might be coordinated.

B) RAR. RAR- together with its heterodimer partner RXR activate the hepatobiliary transporters Ntcp and Mrp2 by binding to retinoid response elements in the promoter regions of these genes. The liver is the major storage site for vitamin A. Retinoids stimulate expression and activity of RXR:RAR-dependent genes such as Ntcp and Mrp2 (81, 345).

C) FXR/bile acid receptors. FXR functions as a heterodimer with RXR and binds with high affinity to inverted repeat response elements (IR-1) where consensus receptor binding hexamers are separated by a single nucleotide. Initial studies demonstrated that induction of the human ileal bile acid binding protein (I-BABP) by bile salts was dependent on FXR/RXR binding to the highly conserved IR-1 (G/A GGTA A TAACCT) (118, 165, 166, 235).

FXR is expressed in tissues where bile salts are transported including the liver, intestine, and kidney and is activated by bile salts with the rank order of potency chenodeoxycholate (CDCA) > deoxycholate (DCA) = lithocholate (LCA) > cholate (CA) (277). Evidence that FXR is the nuclear bile acid receptor (BAR) came from studies where expression plasmids containing murine and human FXR were transfected into monkey kidney CV-1 cells or human hepatoma HepG2 cells and exposed to bile salt metabolites (235). In these experiments CDCA was the most potent activator, with a half-maximal effective concentration (EC₅₀) of 50 and 10 µM for murine and human FXR, respectively. Amino acid residues asparagines (354) and isoleucine (372) confer sensitivity of human FXR to CDCA (75). DCA and LCA also stimulated FXR expression but to a lesser extent, whereas other sterols including cholesterol, oxysteols, steroid hormones, and other bile acid metabolites had no effect (235, 277). Particularly noteworthy was the absence of an activating effect by ursodeoxycholate (UDCA), a commonly used bile acid for the treatment of cholestasis (235), and -muricholic acid, a prominent bile acid that accumulates in the cholestatic rat model (316). Cotransfection assays indicated that FXR/RXR heterodimers were required for bile acids to maximally transactivate the luciferase reporter. In the same studies, FXR was found to repress transcription of the gene encoding for cholesterol 7-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis from cholesterol while stimulating the expression of the human intestinal bile acid binding protein (I-BABP) (235), a 17-kDa protein with high affinity for bile acids, that modulates bile salt uptake in the terminal ileum. Studies in Caco-2 cell lines, derived from human enterocytes, show that bile salts increase I-BABP expression, a finding consistent with the effects of bile diversion and cholystyramine administration in mice where I-Babp expression is diminished (118). Deletion and mutation analyses have established that the FXR/RXR heterodimer activates I-BABP gene expression by binding to bile acid response elements in its promoter as demonstrated in human, mouse, and rabbit (118). Subsequent studies have demonstrated that FXR activates the expression of the transcriptional repressor SHP-1, which functions in a dominant manner to inhibit CYP7A1 in the mouse (see below). Thus bile salts feedback to regulate their own synthesis by binding to FXR and subsequent activation of SHP (Fig. 2). Subsequent studies confirm that both human, rat, and mouse BSEP/Bsep promoters are transcriptionally activated by FXR (13, 110, 287) and that bile salts increase BSEP expression in primary human hepatocytes or HepG2 cells with the same rank order of potency that activates FXR (314). Conversely, LCA decreases BSEP expression by antagonizing FXR activation (396). Recent studies have demonstrated transactivation of Mrp2 (173) and the human OATP8 promoter by FXR (162). In contrast to hepatic bile salt transporters and I-BABP, the ileal sodium-dependent bile salt transporter Isbt was not regulated by FXR, although the studies confirmed a role for FXR in regulation of I-Babp.

In summary, the findings that FXR is abundantly expressed in tissues that express bile salt transporters, that CDCA, DCA, and LCA and their conjugates bind to FXR at physiological concentrations that regulate gene transcription and are highly effective activators of FXR when expressed in cellular expression systems, provide strong evidence that bile salts are the natural ligand for this NHR.

Confirmatory evidence for the importance of FXR in the regulation of expression of several bile salt transporters came from studies with targeted disruption of the nuclear receptor FXR (326). When a 1% cholic acid diet was fed to wild-type mice, no ill effects were observed. Ntcp and CYP7A1 were both downregulated while Bsep was upregulated, thereby limiting the toxic accumulations of bile salts in the liver. In addition, SHP was also upregulated, facilitating downregulation of Ntcp and reducing expression of CYP7A1. However, when th