Apical Sodium-dependent Bile Salt Transporter Research Articles

Administration of ampicillin increases ileal Asbt protein amounts resulting in reduction of fecal bile acid excretion in mice

Administration of the antibacterial drug ampicillin (ABPC) significantly decreased fecal bile acid excretion although it increased hepatic cholesterol 7α‐hydroxylation as well as CYP7A1 mRNA level. In the present study, we have investigated the mechanisms for the reduction of fecal bile acid excretion in ABPC‐treated mice. Bile acid amounts in small intestinal lumen and portal blood concentrations of bile acids were increased 1.6‐fold and 2.0‐fold, respectively, in ABPC‐treated mice. Ileal apical sodium‐dependent bile salt transporter (Asbt) mRNA levels were significantly increased in ABPC‐treated mice whereas ileal bile acid binding protein and organic solute transporter alpha and beta mRNA levels were reduced. Ileal Asbt protein amounts in brush border membranes were increased 2.5‐fold in ABPC‐treated mice. These results suggest that enhancement of ileal Asbt‐mediated bile acid absorption contributes to the reduction of fecal bile acid excretion in ABPC‐treated mice.

Cholesterol Feeding Prevents Hepatic Accumulation of Bile Acids in Cholic Acid-Fed Farnesoid X Receptor (FXR)-Null Mice: FXR-Independent Suppression of Intestinal Bile Acid Absorption

Cholic acid (CA) feeding of farnesoid X receptor (Fxr)-null mice results in markedly elevated hepatic bile acid levels and liver injury. In contrast, Fxr-null mice fed cholesterol plus CA (CA+Chol) do not exhibit liver injury, and hepatic bile acid levels and bile acid pool size are reduced 51 and 40%, respectively, compared with CA-treated Fxr-null mice. These decreases were not observed in wild-type mice. Despite a reduced bile acid pool size, hepatic Cyp7a1 mRNA expression was increased in Fxr-null mice fed the CA+Chol diet, and biliary bile acid output was not changed. Analysis of other potential protective mechanisms revealed significant decreases in portal blood bile acid concentrations and a reduced ileal bile acid absorption capacity, as estimated using an in situ loop method. Fecal bile acid excretion was also increased in Fxr-null mice fed the CA+Chol versus CA diet. The decreased ileal bile acid absorption correlated with decreased ileal apical sodium-dependent bile salt transporter (ASBT) protein expression in brush-border membranes. These results suggest a critical role for ileal bile acid absorption in regulation of hepatic bile acid levels in Fxr-null mice fed CA+Chol. Furthermore, experiments with Fxr-null mice suggest that cholesterol feeding can down-regulate ASBT expression through a pathway independent of FXR.

Novel cationic and neutral glycocholic acid and polyamine conjugates able to inhibit transporters involved in hepatic and intestinal bile acid uptake

To obtain novel drugs able to inhibit transporters involved in bile acid uptake, three compounds were synthesized by conjugating N-(3-aminopropyl)-1,3-propanediamine (PA) with one (BAPA-3), two (BAPA-6), or three (BAPA-8) moieties of glycocholic acid (GC) through their carboxylic group. The expected net charge in aqueous solutions was 2+ (BAPA-3), 1+ (BAPA-6), and 0 (BAPA-8). They were purified by liquid chromatography and their purity checked by HPLC before being chemically characterized by elemental analysis, NMR, and FAB-MS. Using brush-border membranes isolated from rat ileum; their ability to inhibit [ 14C]-GC transport (BAPA-3 > BAPA-6 > BAPA-8) was suggested. This was further investigated 48 h after injecting Xenopus laevis oocytes with the mRNA of rat sodium/taurocholate (TC)-cotransporting polypeptide (Ntcp), rat apical sodium-dependent bile salt transporter (Asbt), or the human isoforms OATP-C/1B1 and OATP8/1B3 of organic anion-transporting polypeptides, when maximal functional expression was detected. BAPA-8, BAPA-6, and BAPA-3 induced no inhibition of OATP8/1B3-mediated [ 3H]-TC uptake, but dramatically reduced [ 3H]-TC uptake by OATP-C/1B1. In the cases of Ntcp- and Asbt-mediated [ 3H]-TC uptake, these were sodium-dependent and were inhibited by BAPA-6 > BAPA-8 > BAPA-3 and BAPA-8 > BAPA-6 > BAPA-3, respectively. In conclusion, our results suggest that these compounds are potentially interesting research tools for the selective modulation of liver and intestinal uptake of bile acids and other cholephilic compounds. Moreover, they may be of pharmacological usefulness to prevent the acute toxicity of compounds reaching liver cells through specific transporters or to enhance both fecal elimination of bile acids and hence cholesterol consumption for the ‘de novo’ synthesis of bile acids.

Apical sodium bile acid transporter and ileal lipid binding protein in gallstone carriers

Although a cholesterol supersaturation of gallbladder bile has been identified as the underlying pathophysiologic defect, the molecular pathomechanism of gallstone formation in humans remains poorly understood. A deficiency of the apical sodium bile acid transporter (ASBT) and ileal lipid binding protein (ILBP) in the small intestine may result in bile acid loss into the colon and might promote gallstone formation by reducing the bile acid pool and increasing the amount of hydrophobic bile salts. To test this hypothesis, protein levels and mRNA expression of ASBT and ILBP were assessed in ileal mucosa biopsies of female gallstone carriers and controls. Neither ASBT nor ILBP levels differed significantly between gallstone carriers and controls. However, when study participants were subgrouped by body weight, ASBT and ILBP protein were 48% and 67% lower in normal weight gallstone carriers than in controls (P < 0.05); similar differences were found for mRNA expression levels. The loss of bile transporters in female normal weight gallstone carriers was coupled with a reduction of protein levels of hepatic nuclear factor 1alpha and farnesoid X receptor. In conclusion, in normal weight female gallstone carriers, the decreased expression of ileal bile acid transporters may form a molecular basis for gallstone formation.

Bile duct proliferation associated with bile salt‐induced hypercholeresis in Mdr2 P‐glycoprotein‐deficient mice

Bile flow consists of bile salt-dependent bile flow (BSDF), generated by canalicular secretion of bile salts, and bile salt-independent flow (BSIF), probably of combined canalicular and ductular origin. Bile salt transport proteins have been identified in cholangiocytes, suggesting a role in control of BSDF and/or in control of bile salt synthesis through cholehepatic shunting. We studied effects of bile duct proliferation under non-cholestatic conditions in multidrug resistance-2 P-glycoprotein (Abcb4)-deficient multidrug resistance gene-2 (Mdr2(-/-)) mice. BSDF and BSIF were determined in wild-type and Mdr2(-/-) mice during infusion of step-wise increasing dosages of tauroursodeoxycholate (TUDC). Cholate synthesis rate was determined by 2H4-cholate dilution. Results were related to expression of transport proteins in liver and intestine. During TUDC infusion, BSDF was increased by approximately 50% and BSIF by approximately 100% in Mdr2(-/-) mice compared with controls. Cholate synthesis rate was unaffected in Mdr2(-/-) mice. Hepatic expression of the apical sodium-dependent bile salt transporter (Asbt), its truncated form (tAsbt) and the multidrug resistance-related protein 3 were upregulated in Mdr2(-/-) mice. Bile duct proliferation in Mdr2(-/-) mice enhances cholehepatic shunting of bile salts, which is associated with a disproportionally high bile flow but does not affect bile salt synthesis.

Partial characterization of cytoprotective mechanisms of lecithin against bile salt-induced bile duct damage

We recently demonstrated that cyclosporine A causes a disproportionate reduction of biliary lipid secretion, and this is inhibited by hydrophilic bile salts through the enhancing of biliary lecithin secretion. In the present study, the underlying mechanism of such a cytoprotective action of hydrophilic bile salts was determined with attention to the possible role of lecithin. Immortalized mouse cholangiocytes were cultured for 4 h with taurine conjugates of a hydrophobic bile salt (cholate [TC]), and hydrophilic bile salts (ursodeoxycholate [TUDC], betamuricholate [TbetaMC], and alphamuricholate [TalphaMC]), at 200 microM, in the presence or absence of lecithin (5, 10, 25, 50, 100, or 200 microM), followed by flow cytometric detection of apoptosis, using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Cholangiocyte bile salt transporter mRNAs (apical sodium-dependent bile-salt transporter [Asbt] and multidrug resistance protein 3 [Mrp3]) were determined by reverse transcription-polymerase chain reaction (RT-PCR). Apoptosis was induced by all of the bile salts (TC > TUDC, TbetaMC, and TalphaMC). Interestingly, bile salt-induced apoptosis was inhibited by lecithin in a concentration-dependent manner. Further, RT-PCR showed that the expressions of Asbt and Mrp3 mRNAs were enhanced by all the bile salts, whereas lecithin reduced Asbt expression, but enhanced Mrp3 expression. These findings indicate that bile salts cause bile-duct cell damage through Asbt-mediated uptake, but that biliary lecithin physiologically inhibits such damage by reducing the expression of this transporter. In addition, the induction of Mrp3 expression by lecithin may play a role in inhibiting the accumulation of bile. Thus, the modulation of lecithin secretion into bile may be another important target for the treatment of biliary disorders.

Enterohepatic bile salt transporters in normal physiology and liver disease

The vectorial transport of bile salts from blood into bile is essential for the generation of bile flow, solubilization of cholesterol in bile, and emulsification of lipids in the intestine. Major transport proteins involved in the enterohepatic circulation of bile salts include the hepatocellular bile salt export pump (BSEP, ABCB11), the apical sodium-dependent bile salt transporter (ASBT, SLC10A2) in cholangiocytes and enterocytes, the sodium-dependent hepatocyte bile salt uptake system NTCP (SLC10A1), the organic anion transporting polypeptides OATP-C (SLC21A6), OATP8 (SLC21A8) and OATP-A (SLC21A3), and the multidrug resistance protein MRP3 (ABCC3). Synthesis and transport of bile salts are intricately linked processes that undergo extensive feedback and feed-forward regulation by transcriptional and posttranscriptional mechanisms. A key regulator of hepatocellular bile salt homeostasis is the bile acid receptor/farnesoid X receptor FXR, which activates transcription of the BSEP and OATP8 genes and of the small heterodimer partner 1 (SHP). SHP is a transcriptional repressor that mediates bile acid-induced repression of the bile salt uptake systems rat Ntcp and human OATP-C. A nuclear receptor that activates rodent Oatp2 (Slc21a5) and human MRP2 (ABCC2) is the pregnane X receptor/steroid X receptor PXR/SXR. Intracellular trafficking and membrane insertion of bile salt transporters is regulated by lipid, protein, and extracellular signal-related kinases in response to physiologic stimuli such as cyclic adenosine monophosphate or taurocholate. Finally, dysfunction of individual bile salt transporters such as BSEP, on account of genetic mutations, steric inhibition, suppression of gene expression, or disturbed signaling, is an important cause of cholestatic liver disease.

Unique inhibition of bile salt-induced apoptosis by lecithins and cytoprotective bile salts in immortalized mouse cholangiocytes.

Bile duct epithelium is physiologically exposed to high concentrations of bile salts, suggesting the presence of a cytoprotective mechanism(s). The aim of this study was to clarify whether bile salts cause bile duct cell damage and to elucidate the mechanism(s) providing protection against such an action of bile salts. Immortalized mouse cholangiocytes were incubated with taurocholate, taurochenodeoxycholate, glycochenodeoxycholate (GCDC), taurodeoxycholate, and tauroursodeoxycholate (TUDC), followed by flow-cytometric analysis and caspase activity assay to evaluate the induction of apoptosis. GCDC time-dependently induced caspase 3 (3.4-fold)- and caspase 9 (1.4-fold)-mediated apoptosis of cholangiocytes, but this was inhibited by lecithins and TUDC. Further, expression of cholangiocyte bile salt transporters (apical sodium-dependent bile salt transporter [Asbt] and multidrug resistance protein 3 [Mrp3]) was examined by RT-PCR and western blotting, and cholangiocyte bile salt uptake was determined using radiolabeled bile salts. Expression of cholangiocyte Asbt and Mrp3 was increased by bile salts, whereas lecithins interestingly reduced bile salt uptake to inhibit cholangiocyte apoptosis. In conclusion, bile salts themselves cause cholangiocyte apoptosis when absorbed by and retained inside the cell, but this is inhibited by washing out cytotoxic bile salts according to Mrp3, a rescue exporting molecule. Biliary lecithin is seemingly another cytoprotective player against cytotoxic bile salts, reducing their uptake, and this is associated with a reduced expression of Mrp3.

Peroxisome Proliferator-activated Receptor α Induces Hepatic Expression of the Human Bile Acid Glucuronidating UDP-glucuronosyltransferase 2B4 Enzyme

Glucuronidation, a major metabolic pathway for a large variety of endobiotics and xenobiotics, is catalyzed by enzymes belonging to the UDP-glucuronosyltransferase (UGT) family. Among UGT enzymes, UGT2B4 conjugates a large variety of endogenous and exogenous molecules and is considered to be the major bile acid conjugating UGT enzyme in human liver. In the present study, we identify UGT2B4 as a novel target gene of the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR alpha), which mediates the hypolipidemic action of fibrates. Incubation of human hepatocytes or hepatoblastoma HepG2 and Huh7 cells with synthetic PPAR alpha agonists, fenofibric acid, or Wy 14643 resulted in an increase of UGT2B4 mRNA levels. Furthermore, treatment of HepG2 cells with Wy 14643 induced the glucuronidation of hyodeoxycholic acid, a specific bile acid UGT2B4 substrate. Analysis of UGT2B mRNA and protein levels in PPAR alpha wild type and null mice revealed that PPAR alpha regulates both basal and fibrate-induced expression of these enzymes in rodents also. Finally, a PPAR response element was identified in the UGT2B4 promoter by site-directed mutagenesis and electromobility shift assays. These results demonstrate that PPAR alpha agonists may control the catabolism of cytotoxic bile acids and reinforce recent data indicating that PPAR alpha, which has been largely implicated in the control of lipid and cholesterol metabolism, is also an important modulator of the metabolism of endobiotics and xenobiotics in human hepatocytes.

The sodium bile salt cotransport family SLC10.

The SLC10 family of sodium/bile salt cotransporters contains over 50 members in animal, plant and bacterial species. In man, two well-characterized members and three orphan transporters are known. The Na(+)/taurocholate cotransporting polypeptide (NTCP; SLC10A1) and the apical sodium-dependent bile salt transporter (ASBT; SLC10A2) are critical components of the enterohepatic circulation of bile salts. NTCP and ASBT are cotransporters that mediate sodium-dependent, electrogenic uptake of mainly bile salts into hepatocytes (NTCP), biliary epithelial cells, ileal enterocytes and renal proximal tubular cells (ASBT).

Cyclosporin a and enterohepatic circulation of bile salts in rats: decreased cholate synthesis but increased intestinal reabsorption.

Cyclosporin A (CsA) has been shown to inhibit synthesis and hepatobiliary transport of bile salts. However, effects of CsA on the enterohepatic circulation of bile salts in vivo are largely unknown. We characterized the effects of CsA on the enterohepatic circulation of cholate, with respect to synthesis rate, pool size, cycling time, intestinal absorption, and the expression of relevant transporters in liver and intestine in rats. CsA (1 mg. 100 g(-1). day(-1) s.c.) or its solvent was administered daily to male rats for 10 days. Cholate synthesis rate and pool size were determined by a 2H4-cholate dilution technique. Bile and feces were collected for determination of cholate and total bile salts, respectively. Cycling time and intestinal absorption of cholate were calculated. The mRNA levels and corresponding transporter protein levels in liver and intestine were assessed by real-time polymerase chain reaction and Western analysis, respectively. CsA treatment decreased cholate synthesis rate by 71%, but did not affect pool size or cycling time. CsA reduced the amount of cholate lost per enterohepatic cycle by approximately 70%. Protein levels of the apical sodium-dependent bile salt transporter (Asbt) were 2-fold increased in distal ileum of CsA-treated rats, due to post-transcriptional events. In conclusion, chronic CsA treatment markedly reduces cholate synthesis rate in rats, but does not affect cholate pool size or cycling time. Our results strongly suggest that CsA enhances efficacy of intestinal cholate reabsorption through increased Asbt protein expression in the distal ileum, which contributes to maintenance of cholate pool size in CsA-treated rats.

Human Apical Sodium-dependent Bile Salt Transporter Gene (SLC10A2) Is Regulated by the Peroxisome Proliferator-activated Receptor α

The apical sodium-dependent bile salt transporter (ASBT/SLC10A2), also called the ileal bile acid transporter, mediates the intestinal absorption of bile salts. The efficiency of this transport process is a determinant of hepatic bile salt synthesis from cholesterol and of serum triglyceride levels. Our aim was to characterize the human ASBT gene promoter with respect to regulatory mechanisms that coordinately affect ASBT expression and hepatic lipid and bile salt metabolism. The minimal construct that confers full promoter activity contains three functional hepatocyte nuclear factor 1alpha (HNF1alpha) recognition sites, explaining the dependence of ASBT gene expression upon HNF1alpha. A nuclear receptor binding site arranged as a direct hexanucleotide repeat (DR1 motif) is localized approximately 1.6 kb upstream of the transcription initiation site. Constructs containing this element were transactivated by WY14643 and ciprofibrate, ligands of the peroxisome proliferator-activated receptor alpha (PPARalpha), in Caco2 cells. The DR1 element was shown to bind the PPARalpha/9-cis-retinoic acid receptor heterodimer, and targeted mutagenesis of the DR1 motif abolished PPARalpha responsiveness. Ciprofibrate treatment of SK-ChA cholangiocytes increased ASBT mRNA levels, suggesting a physiologic role for PPARalpha-mediated ASBT gene regulation. This study identifies PPARalpha as a novel link between ileal bile salt absorption and hepatic lipid metabolism.

Dapsone-induced cholestasis and impairment of bile salt output in the rat

To evaluate the effect of dapsone (4,4'-diaminodiphenylsulfone, DDS) on biliary bile salt secretion, we administered the drug to male and female Wistar rats at a dose of 30 mg/kg body wt, twice a day, for 4 days. DDS decreased basal bile flow by about 20% in both male and female rats. In addition, basal biliary bile salt secretion was decreased by the drug in animals from both sexes (about 30% decrease). Bile salt maximum secretory rate, as evaluated by infusing tauroursodeoxycholate at stepwise-increasing rates, was not affected by DDS in either male or female rats, suggesting that the density of canalicular bile salt transporters is preserved. The size of the bile salt pool and the rate of de novo synthesis of bile salts, measured in bile salt-depleted animals, were decreased by about 33 and 35%, respectively; there was no difference in response between males and females. The ability of the ileum to reabsorb bile salts, as estimated by analysis of the expression of the ileal apical sodium-dependent bile salt transporter and of sodium taurocholate transport activity in brush border membrane vesicles, was not affected by DDS in either males or females. Overall, our findings suggest that an impairment of de novo synthesis mediated by a direct inhibition of CYP3A metabolism, rather than a decreased intestinal reabsorption of bile salts, accounts for the decrease in bile salt pool size. The dissociation between alteration of bile secretory function and the oxidative stress induced by DDS, which is known to be relevant only in male rats, is discussed.

Dapsone-induced cholestasis and impairment of bile salt output in the rat

To evaluate the effect of dapsone (4,4′-diaminodiphenylsulfone, DDS) on biliary bile salt secretion, we administered the drug to male and female Wistar rats at a dose of 30 mg/kg body wt, twice a day, for 4 days. DDS decreased basal bile flow by about 20% in both male and female rats. In addition, basal biliary bile salt secretion was decreased by the drug in animals from both sexes (about 30% decrease). Bile salt maximum secretory rate, as evaluated by infusing tauroursodeoxycholate at stepwise-increasing rates, was not affected by DDS in either male or female rats, suggesting that the density of canalicular bile salt transporters is preserved. The size of the bile salt pool and the rate of de novo synthesis of bile salts, measured in bile salt-depleted animals, were decreased by about 33 and 35%, respectively; there was no difference in response between males and females. The ability of the ileum to reabsorb bile salts, as estimated by analysis of the expression of the ileal apical sodium-dependent bile salt transporter and of sodium taurocholate transport activity in brush border membrane vesicles, was not affected by DDS in either males or females. Overall, our findings suggest that an impairment of de novo synthesis mediated by a direct inhibition of CYP3A metabolism, rather than a decreased intestinal reabsorption of bile salts, accounts for the decrease in bile salt pool size. The dissociation between alteration of bile secretory function and the oxidative stress induced by DDS, which is known to be relevant only in male rats, is discussed.

Bile salt transporters.

Bile salts are the major organic solutes in bile and undergo extensive enterohepatic circulation. Hepatocellular bile salt uptake is mediated predominantly by the Na(+)-taurocholate cotransport proteins Ntcp (rodents) and NTCP (humans) and by the Na(+)-independent organic anion-transporting polypeptides Oatp1, Oatp2, and Oatp4 (rodents) and OATP-C (humans). After diffusion (bound by intracellular bile salt-binding proteins) to the canalicular membrane, monoanionic bile salts are secreted into bile canaliculi by the bile salt export pump Bsep (rodents) or BSEP (humans). Both belong to the ATP-binding cassette (ABC) transporter superfamily. Dianionic conjugated bile salts are secreted into bile by the multidrug-resistance-associated proteins Mrp2/MRP2. In bile ductules, a minor portion of protonated bile acids and monomeric bile salts are reabsorbed by non-ionic diffusion and the apical sodium-dependent bile salt transporter Asbt/ASBT, transported back into the periductular capillary plexus by Mrp3/MRP3 [and/or a truncated form of Asbt (tAsbt)], and subjected to cholehepatic shunting. The major portion of biliary bile salts is aggregated into mixed micelles and transported into the intestine, where they are reabsorbed by apical Oatp3, the apical sodium-dependent bile salt transporter (ASBT), cytosolic intestinal bile acid-binding protein (IBABP), and basolateral Mrp3/MRP3 and tAsbt. Transcriptional and posttranscriptional regulation of these enterohepatic bile salt transporters is closely related to the regulation of lipid and cholesterol homeostasis. Furthermore, defective expression and function of bile salt transporters have been recognized as important causes for various cholestatic liver diseases.

Defects in the enterohepatic circulation of bile acids

ILE ACIDS serve at least two physiological roles, both of which are dependent on their enterohepatic circulation. Bile acid flux is the major driving force of bile production and they are essential for the normal absorption of fats from the gastrointestinal tract. Bile is a mixture of waste products, notably bilirubin and other conjugates, along with bile acids, lipids and inorganic ions, most of which are reabsorbed. Both functions now seem equally important. Whilst bile acids are an essential component of bile they are themselves toxic. The main function of other constituents, notably phospholipids and cholesterol, is to protect against the harmful effects of bile acids, through the formation of mixed micelles. The complete cycle of enterohepatic circulation of bile acids involves transport across two epithelia, as well as passage through the gut and portal blood. Only about 2% of the bile acids excreted daily into the GI tract are lost in the stool. A single transporter appears be responsible for this conservation through uptake in the distal ileum. The apical sodium-dependent bile salt transporter (ASBT) (1,2) is a transmembrane protein similar to the sodiumdependent bile acid transporter found in hepatocytes (NTCP) (3), which is discussed below. The gene is located on chromosome 13q33 and has the symbol SLClOA2 (4). The protein, when expressed in vitro has Ki values in the range of 3.3 PM (chenodeoxycholate) to 41.5 PM (cholate) for bile acids but very high Ki values for a number of other organic anions (5). This contrasts with NTCP, which appears to have a much broader specificity. Recessive, loss of function, mutations in the ASBT gene have been found in patients with primary bile acid malabsorption (4). Bile acids are removed from the portal circulation by hepatocytes. The major bile acid transporter in the basolateral membrane of hepatocytes is the sodium taurocholate co-transporting polypeptide (NTCP) (3). The organic anion transporting polypeptides are capable of transporting bile acids, along with many amphipathic compounds. Whether OATP plays a physiologically significant role in bile acid transport in not clear. Bile acid export from hepatocyes operates against a steep concentration gradient. The biliary bile acid concentration is at least lOOO-fold higher than that in hepatocytes. It is not surprising therefore that the transport of bile acids across this membrane has been shown to be an energy-dependent process. The major transporter is called the bile salt export pump and is encoded by ABCBll (previously called BSEP or SPGP). When the rat cDNA was expressed in vitro, the protein proved to be an effective ATP-dependent bile acid transporter with a Km for taurocholate of -5 ,uM (6). The protein has therefore been named the bile salt export pump. Mutations in the human gene have been found in patients with progressive familial intrahepatic cholestasis with normal levels of serum yGT (7). In those where it has been measured, they also have low levels of biliary bile acids; this correlates well with the low yGT. A similar phenotype has been found in patients with mutations in a P-type ATPase, also located in the hepatocyte canalicular membrane (8). The protein may well be acting as an aminophospholipid flipase. However, it is not clear how mutations in this gene lead to an interruption in the efflux of bile acids from hepatocytes. Most of the major bile acid transporter genes have now been cloned. The intracellular transport of bile acids is less well understood. Recessive mutations have so far been found in three transporter genes, though