ATP-dependent Bile Acid Transport Research Articles

Cloning of the dog bile salt export pump (BSEP; ABCB11) and functional comparison with the human and rat proteins

The dog bile salt export pump (BSEP; ABCB11) was cloned and expressed in a Sf9 insect cell system. The deduced amino acid sequence encodes a 1325-amino-acid protein, which shows 89.4% and 80.2% homology with human BSEP and rat Bsep, respectively. The transcript of the dog Bsep gene was detected at a high level in liver, but not other tissues, by quantitative RT-PCR. The BSEP-expressing membrane vesicles isolated from Sf9 cells exhibited saturable uptake of [(3)H]taurocholic acid with Michaelis constants (K(m)) of 33.7, 22.2 and 19.9 microM for the dog, rat and human transporters, respectively. The uptake of [(3)H]taurocholic acid by all three transporters was significantly inhibited by troglitazone, glibenclamide, and other several inhibitors, while pravastatin inhibited dog Bsep and human BSEP, but not rat Bsep at 100 microM. The IC(50) of troglitazone for dog Bsep, human BSEP, and rat Bsep were 32, 20, and 60 microM, and those of pravastatin were 441, 240 and >1,000 microM, respectively. In conclusion, while dog Bsep shows similar ATP-dependent bile acid transport characteristics to human BSEP and rat Bsep, there is a species difference in affinity for drugs such as pravastatin and troglitazone.

Beneficial effect of ursodeoxycholic acid on alterations induced by cholestasis of pregnancy in bile acid transport across the human placenta

Background/Aims: The existence of impairment in bile acid transport across the placenta during intrahepatic cholestasis of pregnancy and the effect of ursodeoxycholic acid treatment (1 g/day) were investigated. Methods: Kinetic parameters were calculated from experiments carried out on membrane vesicles obtained from basal (TPMb, fetal-facing) and apical (TPMa, maternal-facing) trophoblast plasma membranes. Bile acid uptake was measured using varying concentrations of [ 14C]-glycocholate and a rapid filtration technique. Results: The maximal velocity of transport (Vmax), the apparent affinity constant (Kt) and the efficiency (Ef) of transport (Vmax/Kt) of the anion:bile acid exchanger located at the TPMb were reduced in intrahepatic cholestasis of pregnancy. Ursodeoxycholic acid induced a reversal of Vmax, Kt and Ef to normal values. Owing to the 3-fold increase in Vmax, with no change in Kt, intrahepatic cholestasis of pregnancy induced an enhancement in Ef of ATP-independent bile acid transport across TPMa. Both Vmax and Ef were restored to normal values by ursodeoxycholic acid. Finally, in ATP-dependent bile acid transport across TPMa, a reduction in the Ef due to an increase in Vmax together with a more pronounced increase in Kt was found. This impairment was also reversed by ursodeoxycholic acid. Conclusions: These results suggest that placenta bile acid transport systems are impaired in intrahepatic cholestasis of pregnancy. Moreover, together with the confirmed beneficial effect for intrahepatic cholestasis of pregnancy patients, such as the relief of pruritus and the improvement in biochemical markers of cholestasis, ursodeoxycholic acid treatment restores the ability of the placenta to carry out vectorial bile acid transfer.

Effect of maternal cholestasis on the kinetics of bile acid transport across the canalicular membrane of infant rat livers.

A reversible impairment in the ability of the liver to secrete cholephilic compounds has been reported to exist in infant rats born from mothers with surgically induced complete cholestasis during the last third of the pregnancy. Canalicular plasma membranes (CPM) were purified from livers obtained from 4 and 8 week-old offspring of healthy or cholestatic rats. Using radiolabelled glycocholic acid (GC) and a rapid filtration technique, bile acid transport by CPM vesicles in the presence of 3 mM ATP plus an ATP-regenerating system was measured at varying substrate concentrations. Kinetic parameters were calculated by nonlinear regression analysis. Similar values for the apparent affinity constant (Kt) were found in all experimental groups (approximately 350 microM). The value of the maximal velocity of the transport (Vmax) was similar for CPM obtained from control animals at 4 or 8 weeks of age (approximately 1.5 nmol/20 s/mg protein). In the offspring of cholestatic mothers the Vmax value was not different from that found in control animals as far as 4 week-old rats were concerned. However, Vmax in the 8 week-old group from cholestatic mothers was two-fold higher than that found in the rest of the experimental groups. Thus, the efficiency of transport, defined as Vmax/Kt, was very similar in all experimental groups, except in the group of 8 week-old offspring of cholestatic mothers, where this value was 60% higher. Isolated livers obtained from this group were able to secrete a tracer dose of radiolabelled GC (11.25 nmol) into bile significantly faster than isolated livers obtained from control animals of the same age (8 weeks). In sum, these results indicate that, in young infant rats (4 week-old), in which the maximal secretion rate for bile acids was reduced by maternal cholestasis during pregnancy, the kinetics of ATP-dependent bile acid transport across the canalicular membrane were not affected. By contrast, in older infant rats (8 week-old), in which the overall ability of the liver to secrete bile acids seems to be restored to normality, the efficiency of the canalicular transport system was actually enhanced. This suggests the existence of compensation at the level of the canalicular membrane transfer and thus that there is another hitherto unidentified mechanism involved in bile acid secretion.

Evidence for an ATP-dependent bile acid transport protein other than the canalicular liver ecto-ATPase in rats

BACKGROUND & AIMS: Canalicular secretion is rate limiting in overall blood-to-bile transport of bile acids. Studies using transfected cells have implicated the canalicular ecto-adenosine triphosphatase (ecto- ATPase) in adenosine triphosphate (ATP)-dependent bile acid transport. However, the structural features of this ecto-ATPase are not those anticipated for an in-to-out ATP-dependent transporter. The aim of this study was to explore the possible existence of an ATP-dependent bile acid transport mechanism distinct from ecto-ATPase. METHODS: Bile acid transport activity and ecto-ATPase expression were analyzed in primary rat hepatocytes, rat hepatoma HTC cells, and specially adapted HTC (HTC- R) cells using plasma membrane vesicles and Northern blot, slot blot, ribonuclease protection assay, and Western blot analyses. RESULTS: Plasma membranes isolated from HTC-R cells exhibited ATP-dependent taurocholate transport, which was many-fold greater than that in HTC cells. Hepatocytes showed the highest transport rates. Protein and RNA analyses showed very low expression of ecto-ATPase in HTC and HTC-R cells compared with hepatocytes. There was no difference between the two cell types at both the RNA and protein level. CONCLUSIONS: These findings show the presence in HTC-R cells and, apparently in hepatocytes, of one or more proteins other than the ecto-ATPase that mediate ATP-dependent transport of bile acids. (Gastroenterology 1997 Jul;113(1):249-54)

A Yeast ATP-binding Cassette-type Protein Mediating ATP-dependent Bile Acid Transport

ATP-dependent transport of bile acids is a key determinant of bile flow in mammalian liver and is associated with cholesterol excretion, gallstone formation, and numerous inherited and acquired hepatobiliary diseases. Secretory vesicles and a vacuole enriched fraction purified from Saccharomyces cerevisiae also exhibit ATP-dependent bile acid transport. ATP-dependent transport of bile acids by the vacuolar fraction was independent of the vacuolar proton ATPase, responded to changes in the osmotically sensitive intravesicular space, and was saturable, exhibiting a Km of 63 microM for taurocholate. The BAT1 (bile acid transporter) gene was isolated from yeast DNA by polymerase chain reaction amplification using degenerate oligonucleotides hybridizing to conserved regions of ABC-type proteins. ATP-dependent bile acid transport was abolished when the BAT1 coding region was deleted from the genome and restored upon reintroduction of the gene. The deduced amino acid sequence predicts that Bat1p is an ABC-type protein 1661 amino acids in length, similar to mammalian cMOAT/cMRP1 and MRP1 transporters, yeast Ycf1p, and two yeast proteins of unknown function. Information obtained from the yeast BAT1 gene may aid identification of the gene encoding the mammalian bile acid transporter.

Differential interaction of bile acids from patients with inborn errors of bile acid synthesis with hepatocellular bile acid transporters.

People with genetic or acquired defects in the biosynthesis of bile acids may suffer from cholestasis. Patients with a deficiency of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase from 3 beta, 7 alpha-dihydroxy- and 3 beta, 7 alpha, 12 alpha-trihydroxy-5-cholenoic acids, the sulfated and partially glycine-conjugated forms of which are found in their urine and bile. 3-Oxo-delta 4 bile acids are detected in the urine of patients with a deficiency of 5 beta-reductase. It has been postulated that these unusual bile acids might act as cholestatic agents in these patients. The aim of the present study was to test this hypothesis in an in vitro system, since the abnormal bile acids would be metabolized in in vivo experiments. Basolateral (sinusoidal) and canalicular plasma membrane vesicles were isolated from rat liver. A rapid filtration method was used to determine transport of cholyltaurine in the presence of model bile acids into the isolated vesicles. It was found that 3 beta, 7 alpha-dihydroxy-5-cholenoic acid and 7 alpha-hydroxy-3-oxo-4-cholenoic acid both inhibited the apical, ATP-dependent transport system for cholyltaurine in a competitive manner with K(m) values of 15 microM and 16 microM, respectively. Radioactively labeled 3 beta, 7 alpha-dihydroxy-5-cholenoyltaurine and 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine were not transported by the same transport system. The same types of experiments were performed with basolateral plasma membrane vesicles. It was found that, in contrast to the canalicular ATP-dependent bile acid transport system, only 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine was a competitive inhibitor of the sodium-dependent transport system for cholyltaurine with a K(m) of 16 microM. Studies with radioactively labeled 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine and 3 beta, 7 alpha-dihydroxy-5-cholenoyltaurine revealed that 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine was transported in a sodium-dependent manner into basolateral rat liver plasma membrane vesicles, whereas 3 beta, 7 alpha-dihydroxy-5-cholenoyltaurine was not transported in a sodium-dependent way. These results support the hypothesis that the unusual bile acids found in patients with defects in bile acid biosynthesis might act as cholestatic agents by inhibiting the canalicular ATP-dependent transport system for bile acids which constitutes the rate-limiting step in the overall process of bile acid transport across hepatocytes. Furthermore, the experiments demonstrated that, despite similar substrate specificities, the basolateral sodium-dependent and the apical ATP-dependent transport system for cholyltaurine might have different recognition sites for bile acids.

ATP-dependent transport of beta-estradiol 17-(beta-D-glucuronide) in rat canalicular membrane vesicles.

The ATP-dependent transport of beta-estradiol 17-(beta-D-glucuronide) (E217G), a cholestatic metabolite of estradiol, was investigated in rat liver canalicular membrane vesicles. ATP-dependent transport was dependent on time and temperature and occurred into an osmotically sensitive space; kinetic analysis indicated a saturable transport system (Michaelis-Menten constant value, 75 microM; maximum transport rate, 598 pmol.min-1.mg protein-1). The steroid conjugates estradiol glucuronide, estriol 3-glucuronide, estriol 16 alpha-glucuronide, testosterone glucuronide, and the three-sulfate conjugate of 17G were effective inhibitors of transport. Bromosulfophthalein, S-(2,4-dinitrophenyl)glutathione, and glutathione disulfide, all substrates of the canalicular ATP-dependent non-bile acid organic anion transport system, were also effective inhibitors, whereas taurocholate had no effect on transport. Conversely, E217G inhibited the ATP-dependent transport of S-(2,4-dinitrophenyl)glutathione. Daunorubicin, vinblastine, etoposide, cyclosporin, and PSC-833, substrates/modulators of P-glycoprotein, were also potent inhibitors of E217G transport, and E217G competitively inhibited the ATP-dependent transport of daunorubicin. C219, a monoclonal antibody against P-glycoprotein, inhibited ATP-dependent transport of E217G and daunorubicin but not of taurocholate or S-(2,4-dinitrophenyl)glutathione. These data indicate that E217G is substrate of both the non-bile acid organic anion transport system and P-glycoprotein but not of the ATP-dependent bile acid transport system in canalicular membranes.

Reconstitution and characterization of ATP-dependent bile acid transport in human and rat placenta.

Bile acid (BA) transport across the human microvillus maternal-facing trophoblast plasma membrane (mTPM) has been recently reported to be stimulated by the presence of ATP [Marin, Bravo, El-Mir and Serrano (1993) J. Hepatol. 18, S41]. Reconstitution of BA transport activity in proteoliposomes from human mTPM is reported in this paper. Typical characteristics of BA transport in native mTPM vesicles, including a requirement for ATP hydrolysis and inhibition by other BA species, were preserved in proteoliposome preparations. BA transport into 20- and 14-day-gestation rat mTPM vesicles was also stimulated by the presence of ATP as noted in human mTPM and in the rat liver canalicular membrane. Besides this functional similarity, these ATP-dependent carriers may share structural characteristics, as demonstrated by studies using an antibody (100 Ab) raised against the 100 kDa BA carrier of the canalicular membrane from rat liver which recognized proteins in both human and rat brush-border trophoblast membranes.

ATP-dependent bile acid transport across microvillous membrane of human term trophoblast

The main fate for fetal bile acids is to be transferred to the mother by the trophoblast. In this study, ATP-dependent bile acid transport across the maternal- and the fetal-facing plasma membranes (mTPM and fTPM, respectively) of the human trophoblast was investigated. With the use of [14C]glycocholate (GC) and a rapid-filtration technique, GC transport by mTPM and fTPM was measured in the absence or the presence of 3 mM ATP plus an ATP-regenerating system. GC efflux from preloaded mTPM or fTPM vesicles was found to be insensitive to ATP. By contrast, GC uptake by mTPM, but not by fTPM, was significantly increased (approximately threefold) by ATP. This was temperature sensitive and occurred into an osmotically reactive space. Kinetic analysis revealed that GC uptake by mTPM was saturable and fit the Michaelis-Menten equation both in the absence and in the presence of ATP. ATP-dependent transport was not abolished by a protonophore (carbonyl cyanide p-trifluormethoxyphenyl hydrazone) together with 100 mM K+ (in = out) plus a K+ ionophore (valinomycin). It specifically required hydrolyzable ATP, although CTP had a slight stimulatory effect. Neither Na+ nor Cl- (100 mM, in = out) was mandatory. Moreover, 100 mM gradients of either Na+ (in << out) or Cl- (in >> out) had no effect on ATP-dependent GC uptake. This was inhibited by vanadate and bile acid analogues but not by several cholephilic organic anions and a variety of adenosine triphosphatase inhibitors. These results provide strong evidence for the existence of an ATP-dependent transport system for bile acids across the apical membrane of human trophoblast, which may play an important role in the control of the overall fetal-maternal bile acid traffic.

Structure-specific inhibition by bile acids of adenosine triphosphate—dependent taurocholate transport in rat canalicular membrane vesicles

The adenosine triphosphate (ATP)-dependent transport system is a major determinant of canalicular bile acid secretion. The system transports bile acids and neither organic cations nor non—bile acid organic anions, such as glucuronides or glutathione adducts. To define the structural specificity of the ATP-dependent system, the authors examined the ability of various bile acids to inhibit ATP-dependent taurocholate transport by rat liver canalicular membrane vesicles. Only bile acids with a negative charge inhibited transport, which was unaffected by side chain length. Conjugated, but not unconjugated, mono- and di-hydroxy bile acids inhibited transport. The presence of 7α- and 12α-hydroxylation also influenced inhibition of ATP-dependent taurocholate transport. Inhibition of transport by bile acids was kinetically competitive. These results suggest that the canalicular ATP-dependent bile acid transport system depends on bile acid side chain charge, conjugation, and hydroxylation.

Structure-specific inhibition by bile acids of adenosine triphosphate—dependent taurocholate transport in rat canalicular membrane vesicles

The adenosine triphosphate (ATP)-dependent transport system is a major determinant of canalicular bile acid secretion. The system transports bile acids and neither organic cations nor non-bile acid organic anions, such as glucuronides or glutathione adducts. To define the structural specificity of the ATP-dependent system, the authors examined the ability of various bile acids to inhibit ATP-dependent taurocholate transport by rat liver canalicular membrane vesicles. Only bile acids with a negative charge inhibited transport, which was unaffected by side chain length. Conjugated, but not unconjugated, mono- and di-hydroxy bile acids inhibited transport. The presence of 7 alpha- and 12 alpha-hydroxylation also influenced inhibition of ATP-dependent taurocholate transport. Inhibition of transport by bile acids was kinetically competitive. These results suggest that the canalicular ATP-dependent bile acid transport system depends on bile acid side chain charge, conjugation, and hydroxylation.

Hepatocellular transport of bile acids. Evidence for distinct subcellular localizations of electrogenic and ATP-dependent taurocholate transport in rat hepatocytes.

To investigate whether electrogenic and ATP-dependent taurocholate transport activities are both mediated by the same bile acid-transporting polypeptide in rat liver, we further purified isolated canalicular membrane vesicles by free flow electrophoresis. Removal of most of the contaminating endoplasmic reticulum resulted in a complete loss of electrogenic taurocholate transport from an ecto-ATPase-enriched canalicular membrane subfraction. In contrast, ATP-dependent taurocholate transport remained associated with both an ecto-ATPase-enriched and an ecto-ATPase-free canalicular membrane subfraction. Microsomes containing 64% of total endoplasmic reticulum exhibited saturable electrogenic (Km approximately 270 microM), but no ATP-dependent taurocholate uptake. Golgi membrane vesicles were devoid of any taurocholate transport activity. These results indicate that electrogenic taurocholate transport resides entirely in the endoplasmic reticulum, whereas ATP-dependent bile acid transport is an intrinsic function of the canalicular membrane as well as of a so far unidentified intracellular membrane bound compartment. Hence, the two transport activities are most probably mediated by two different bile acid transporting polypeptides. Furthermore, the finding of ATP-dependent taurocholate transport in virtually ecto-ATPase-free vesicles argues against the concept of primary active bile acid transport being exclusively mediated by the canalicular ecto-ATPase.