Bile Acid Transport System Research Articles

First-pass elimination of a peptidomimetic thrombin inhibitor is due to carrier-mediated uptake by the liver: Interaction with bile acid transport systems

CRC 220 (4-methoxy-2, 3, 6-trimethylphenylsulfonyl-L-aspartyl-D-4-amidinophenylalanyl-piperidide) is a competitive peptide-based trombin inhibitor with high affinity to human α-thrombin ( K i 2.5 nM). The amphiphilic compound exhibits virtually no systemic bioavailability despite proteolytic stability and proven enteral absorption. After intravenous application (V. jejunalis) in rats CRC 220 is almost completely excreted into bile. Simultaneous administration of bile acids considerably decreases this first-pass elimination. CRC 220 is extensively taken up in isolated rat hepatocytes by a saturable carrier-mediated transport with K m 23.7 μM and V max 775 pmol × mg −1 × min −1. A large part of this transport is energy-dependent. At temperatures above 20 °C, the uptake is accelerated exponentially. The activation energy amounts to 82 kJ/mol. A minor portion of CRC 220 uptake occurs by physical diffusion with a permeability coefficient of 7.83 × 10 −7cm/sec at 12 °C. Sodium ions energize CRC 220 uptake. Replacement of sodium by choline or lithium decreases the transport rate of 23–40%. In addition, a negative membrane potential facilitates the uptake. CRC 220 transport is only observed in hepatocytes: it is absent in BHK, FAO, HepG2, HPCT 1E3, and HPCT 1E3-TC cells. In the presence of 4-amidinophenylalanine derivatives, CRC 220 uptake is considerably decreased. Inhibition also occurs with bile acids and bromosulfophthalein, but less with bumetanide. Because CRC 220 inhibits bile acid uptake into hepatocytes and vice versa, the results suggest that the first-pass elimination of this amphiphilic thrombin inhibitor is due to an active carrier-mediated transport process in the basolateral plasma membrane of rat hepatocytes, and that this transport occurs via a bile acid transport system.

Bumetanide is not transported by the Ntcp or by the oatp: evidence for a third organic anion transporter in rat liver cells

The loop diuretic bumetanide which inhibits hepatic bile acid uptake competitively according to its transport kinetics has been proposed to serve as a substrate of a multispecific bile acid transport system in liver parenchymal cells. However, when the in vitro transcripts of two cloned hepatic bile acid uptake carriers, the Ntcp (Na +/taurocholate cotransporting polypeptide) and the oatp (organic anion transporting polypeptide), was expressed for three days in Xenopus laevis oocytes [ 3H]bumetanide uptake was not increased although bile acid uptake was stimulated. The data presented show that bumetanide is taken up by a third organic anion transport system which is different from the cloned bile acid transporters.

Transport of dehydroepiandrosterone and dehydroepiandrosterone sulphate into rat hepatocytes

The purpose of the present study was to characterize the transport of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) into hepatocytes at physiological and pharmacological concentrations. Hepatocytes were isolated from female Sprague-Dawley rats by collagenase perfusion. Uptake of [ 3H]DHEA and [ 3H]DHEAS at increasing concentrations (3.5 nM-100 μM) was measured by the rapid filtration technique at 30 s intervals up to 120 s. The uptake of DHEAS by hepatocytes was saturable ( K m = 17.0 μM; V max = 3.7 nmol/min/mg cell protein). In contrast, a specific saturable transport system for DHEA could not be detected in rat hepatocytes. It is suggested that DHEA enters the cell by diffusion. The uptake of DHEAS could be inhibited by antimycin A, carbonylcyanide- m-chlorophenylhydrazone, and dinitrophenol (inhibitors of the mitochondrial respiratory chain), by dinitrofluorobenzene and p-hydroxymercuribenzoate (NH 2- and SH-blockers, respectively), and by monensin (Na +-specific ionophore). No inhibition was seen in the presence of ouabain (inhibitor of Na +-K +-ATPase) and phalloidin (inhibitor of cholate transport and actin-blocker). Interestingly, DHEAS uptake was inhibited by bile acids (cholate, taurocholate and glycocholate). Conversely, [ 3H]cholate uptake was strongly inhibited by DHEAS, which indicates a competition for the same carrier. Replacement of sodium ion with choline markedly decreased uptake velocity at pharmacological DHEAS concentrations. The results suggest that DHEAS uptake is a saturable, energy-dependent, carrier-mediated, partially Na +-dependent process, and that DHEAS may be taken up via the multispecific bile acid transport system.

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.

Hepatic uptake of octreotide, a long-acting somatostatin analogue, via a bile acid transport system.

The hepatic transport mechanism of octreotide (Sandostatin), a somatostatin analogue, was studied using freshly prepared rat hepatocytes. The initial uptake rate of octreotide represented exclusively a saturable transport process. The half-saturation constant, Kt, and the maximum uptake-rate, Jmax, for the uptake of octreotide were 91.1 +/- 28.4 microM and 104.6 +/- 19.7 pmol/mg protein/min, respectively. An energy requirement was demonstrated for [14C]octreotide uptake since metabolic inhibitors (DNP, rotenone, antimycin and NaCN) significantly reduced the initial uptake rate. [14C]octreotide uptake was also significantly inhibited by ouabain. [14C]octreotide uptake was reduced in the absence of Na+ in the uptake medium. [14C]octreotide uptake was significantly inhibited by bile acids, iodipamide, d-tubocurarine, whereas it was not inhibited by bilirubin, TEMA and insulin. Competitive inhibition of taurocholic acid was observed for octreotide uptake with the inhibition constant, Ki, of 82 +/- 17 microM. Moreover, a significant inhibitory effect of octreotide was observed for the Na+ dependent uptake of [14C]taurocholic acid. These results suggest that octreotide is transported into hepatocytes via a bile acid carrier-mediated system.

Synthesis and biological activity of bile acid-derived HMG-CoA reductase inhibitors. The role of 21-methyl in recognition of HMG-CoA reductase and the ileal bile acid transport system.

To increase hepatoselectivity of HMG-CoA reductase inhibitors by using the specific bile acid transport systems, deoxycholic acid-derived inhibitors 9 and 11 have been synthesized, on the basis of the concept of combining in one molecule structural requirements for specific inhibition of the HMG-CoA reductase and specific recognition by the ileal bile acid transport system. The 1-methyl-3-carboxylpropyl subunit of deoxycholic acid was replaced by the 3,5-dihydroxyheptanoic acid lactone of lovastatin, and position 12-OH was esterified with 2-methylbutyric acid. Compounds 9 and 11 were evaluated for their inhibitory activity on rat liver HMG-CoA reductase, cholesterol biosynthesis in HEP G2 cells, and [3H]taurocholate uptake in rabbit brush border membrane vesicles and compared with methyl derivatives 8 and 10. The steroidal 21-CH3 group affects both activity on HMG-CoA reductase and recognition by the ileal bile acid transport system.

Binding proteins for cyclosomatostatins and bile acids in basolateral plasma membranes of rat liver

The bile acids cholate and taurocholate on the one hand and the cyclopeptide c(Phe-Thr-Lys-Trp-Phe-D-Pro) (008), an analog of somatostatin with retro sequence, on the other hand, display mutually competitive transport inhibition into isolated rat hepatocytes. This indicates a common transport system for bile acids and cyclosomatostatins in sinusoidal rat liver plasma membranes. In order to identify and isolate common binding and/or transport proteins for bile acids and the cyclopeptides by affinity chromatography, the bile acid derivative 4′-amino-7-benzamidotaurocholate (ABATC) and the cyclosomatostatin-analog 008 were attached to a gel matrix. Two methods were used to prepare integral membrane proteins: (1) alkaline EDTA extraction and (2) Triton X-114 phase separation. Octyl glycoside solubilized, alkaline EDTA-extracted integral basolateral membrane proteins with apparent molecular masses of 52 and 48 kDa bound specifically to the ABATC affinity matrix. Two-phase Triton X-114 separated integral membrane proteins of the same molecular masses bound specifically to the cyclosomatostatin ligand. The 48 kDa ABATC and 008 binding protein was shown to be present in the basolateral plasma membrane fraction and in the microsomal fraction. The isolated 52 kDa ABATC binding protein was localized only in basolateral plasma membranes and could not be found in isolated microsomes.

Intestinal absorption of peptides by coupling to bile acids.

Poor intestinal absorption of peptides greatly limits their use as drugs for the treatment of chronic diseases. Since bile acids are efficiently absorbed by an active, Na(+)-dependent transport system in the ileum of mammals, model peptides of different chain length were attached to the 3-position of modified 3 beta-(omega-amino-alkoxy)-7 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid. These peptide-bile acid conjugates inhibited Na(+)-dependent [3H]taurocholate uptake into brush-border membrane vesicles isolated from rabbit ileum in a concentration-dependent manner. Furthermore, photoaffinity labeling of the bile acid-binding proteins of M(r) 93,000 and 14,000, identified as the protein components of the ileal Na(+)-dependent bile acid transport system in rabbit ileum (Kramer, W., Girbig, F., Gutjahr, U., Kowalewski, S., Jouvenal, K., Müller, G., Tripier, D., and Wess, G. (1993) J. Biol. Chem. 268, 18035-18046) by the photoreactive taurocholate analogue, (3,3-azo-7 alpha, 12 alpha-dihydroxy-5 beta [7 beta, -12 beta-3H]cholan-24-oyl)-2-aminoethanesulfonic acid, was inhibited by the peptide-bile acid conjugates. In contrast, the parent peptides and amino acids neither had a significant effect on [3H]taurocholate uptake by ileal brush-border membrane vesicles nor on photoaffinity labeling of the ileal bile acid-binding membrane proteins. The inhibitory effect of peptide-bile acid conjugates on [3H]taurocholate transport and photoaffinity labeling of the bile acid-binding proteins in rabbit ileal vesicles decreased with increasing chain length of the attached peptide radical. By in vivo ileum perfusion in anesthetized rats an intestinal absorption of the bile acid conjugate S3744 of the fluorescent oxaprolylpeptide 4-nitrobenzo-2-oxa-1,3-diazol-beta-Ala-Phe-5-Opr-Gly (S1037) and secretion of the intact compound into bile could be demonstrated, whereas the parent peptide S1037 or its t-butylester S4404 were not absorbed. The intestinal absorption of S3744 showed a similar temperature dependence as [3H]taurocholate absorption and was inhibited by the presence of taurocholate indicating a carrier-mediated uptake of S3744 via the ileal bile acid transporter. In conclusion, these results indicate that oligopeptides can be made enterally absorable by coupling to modified bile acid molecules making use of the specific intestinal absorption pathway for bile acids. This finding may be of great importance for the design and development of orally active peptide drugs.

Expression cloning and characterization of the hamster ileal sodium-dependent bile acid transporter.

Active uptake of bile acids from the lumen of the small intestine is mediated by an ileal Na(+)-dependent bile acid transport system. To identify components of this transport system, an expression cloning strategy was employed to isolate a hamster ileal cDNA that exhibits bile acid transport activity. By Northern blot analysis, mRNA for the cloned transporter was readily detected in ileum and kidney but was absent from liver and proximal small intestine. The transporter cDNA encoded a 348-amino acid protein with seven potential transmembrane domains and three possible N-linked glycosylation sites. The amino acid sequence was 35% identical and 63% similar to the rat liver Na+/bile acid cotransporter. After transfection into COS cells, the hamster cDNA transported taurocholate in a strict Na(+)-dependent fashion with an apparent Km of 33 microM. This taurocholate transport was inhibited by various bile acids but not by taurine or other organic anions. The Na+ dependence, saturability, and bile acid specificity of transport as well as the tissue specificity of mRNA expression strongly argue that the transporter cDNA characterized in this study is the Na+/bile acid cotransporter described previously in ileum.

Developmental regulation of hepatic bile acid transport systems , Department of Pediatrics and the Liver Center, Yale University, New Haven, CT, 06510

Developmental regulation of hepatic bile acid transport systems , Department of Pediatrics and the Liver Center, Yale University, New Haven, CT, 06510

Synthesis of bile acid - drug conjugates: Potential drug - shuttles for liver specific targeting

Cholic acid ( 1) has been coupled via ω-aminoalkoxy spacer at C-3 to chlorambucil ( 3), HR 780 ( 4) and oxaproline peptide ( 5). Drug conjugates 9, 11, 18 exhibit strong affinity to specific bile acid transport systems.

Negative feedback regulation of the ileal bile acid transport system in rodents

Background: Active transport of conjugated bile acids by ileal enterocytes is a key mechanism for conservation of the bile acid pool. Experiments were performed to determine whether such transport is regulated by substrate load. Methods: Using anesthetized biliary fistula guinea pigs or rats, the ileum was perfused with ursodeoxycholyltaurine at a concentration causing maximal ileal transport of this bile acid; absorption was assessed by biliary recovery. Before ileal perfusion, animals ingested one of three diets: chow, chow with added conjugated bile acid, or chow with added cholestyramine. Results: In the guinea pig, ingestion of a taurocholate-enriched diet resulted in a 75% decrease in the absorption rate of ursodeoxycholyltaurine. Similar results were obtained with cholylsarcosine (a deconjugation-dehydroxylation resistant analogue) or with chenodeoxycholylglycine, the endogenous bile acid of the guinea pig. In contrast, cholestyramine ingestion caused an increase in ursodeoxycholyltaurine absorption. In the rat, cholyltaurine or cholylsarcosine ingestion also caused decreased ileal transport. In the guinea pig, maximal down-regulation of active ileal bile acid transport occurred after 2–3 days of bile acid feeding; up-regulation required 3–4 days. Conclusions: Bile acid metabolism is regulated by feedback inhibition of active ileal transport in addition to the well-established feedback inhibition of bile acid biosynthesis in the liver. Together, these two regulatory mechanisms ensure constancy of bile acid secretion.

Characterization and chemical modification of the Na +-dependent bile-acid transport system in brush-border membrane vesicles from rabbit ileum

The Na 1-dependent uptake system for bile acids in the ileum from rabbit small intestine was characterized using brush-border membrane vesicles. The uptake of [ 3H]taurocholate into vesicles prepared from the terminal ileum showed an overshoot uptake in the presence of an inwardly-directed Na +-gradient ([Na +] out > [Na +] in), in contrast to vesicles prepared from the jejunum. The Na +-dependent [ 3H]taurocholate uptake was cis-inhibited by natural bile acid derivatives, whereas cholephilic organic compounds, such as phalloidin, bromosulphophthalein, bilirubin, indocyanine green or DIDS — all interfering with hepatic bile-acid uptake — did not show a significant inhibitory effect. Photoaffinity labeling of ileal membrane vesicles with 3,3-azo- and 7,7-azo-derivatives of taurocholate resulted in specific labeling of a membrane polypeptide with apparent molecular mass 90 kDa. Bile-acid derivatives inhibiting [ 3H]taurocholate uptake by ileal vesicles also inhibited labeling of the 90 kDa polypeptide, whereas compounds with no inhibitory effect on ileal bile-acid transport failed to show a significant effect on the labeling of the 90 kDa polypeptide. The involvement of functional amino-acid side-chains in Na +-dependent taurocholate uptake was investigated by chemical modification of ileal brush-border membrane vesicles with a variety of group-specific agents. It was found that (vicinal) thiol groups and amino groups are involved in active ileal bile-acid uptake, whereas carboxyl- and hydroxyl-containing amino acids, as well as tyrosine, histidine or arginine are not essential for Na +-dependent bile-acid transport activity. The irreversible inhibition of [ 3H]taurocholate transport by DTNB or NBD-chloride could be partially reversed by thiols like 2-mercaptoethanol or DTT. Furthermore, increasing concentrations of taurocholate during chemical modification with NBD-chloride were able to protect the ileal bile-acid transporter from inactivation. These findings suggest that a membrane polypeptide of apparent M r 90 000 is a component of the active Na +-dependent bile-acid reabsorption system in the terminal ileum from rabbit small intestine. Vicinal thiol groups and amino groups of the transport system are involved in Na +-dependent transport activity, whereas other functional amino acids are not essential for transport activity.

Tissue-selective action of pravastatin due to hepatocellular uptake via a sodium-independent bile acid transporter

Pravastatin is foreign substrate of a sodium-independent transport system for bile acids. The tissue selectivity of pravastatin in inhibiting 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase is due to the uptake via a transport system which exists predominantly in liver cells. Pravastatin competitively inhibits the sodium-independent hepatocellular uptake of cholate, taurocholate and ouabain, whereas the total uptake of cholate is non-competitively blocked. The affinity of pravastatin to the sodium-dependent taurocholate transporter is, however, low. Millimolar concentrations of pravastatin are needed to inhibit the sodium-taurocholate cotransporter. Pravastatin has no affinity to other transport systems in liver cells such as those for long-chain fatty acids, amino acids, rifampicin and bivalent organic cations.

Modified bile acids: preparation of 7α,12α-dihydroxy-3β- and 7α,12α-dihydroxy-3α-(2-hydroxyethoxy)-5β-cholanic acid and their biological activity

Methodology for the preparation of 7α-12α-dihydroxy-3β- (2) and 7α,12α-dihydroxy-3α-(2-hydroxyethoxy)-5β-cholanic acid (3) is described. Nucleophilic displacement of the 3-mesylate of unprotected cholic acid with ethylene glycol led to the 3β-isomer whereas the 3α-isomer was synthesized via the 7,12-diacetyl protected 3-allyl ether of methyl cholate. Only the 3α-isomer 3 is recognized by the ileal bile acid transport system with affinity comparable to cholic acid.

Functional molecular mass of the 14C-azidobenzamidotaurocholic acid binding proteins in hepatocellular bile acid transport systems

The apparent target size of 14C-azidobenzamidotaurocholate binding proteins in basolateral rat liver plasma membranes (BlPm) was determined by analysis of the radiation induced decrease of the binding of this photoreactive taurocholate analog to blPm. Radiation causes a dose-dependent mono-exponential reduction of binding of ABATC to the protein subunits with molecular masses of 48–50 and 52–54 kDa in SDS-PAGE. The minimal functional molecular mass of the 48–50 and 52–54 kDa ABATC binding proteins was determined to be 99±8.2 and 93.2±7 kDa, respectively.

An ontogenically regulated 48-kDa protein is a component of the Na(+)-bile acid cotransporter of rat liver

Recent evidence suggests that the Na(+)-coupled carrier mechanism for bile acids on the hepatocyte basolateral plasma membrane is a polypeptide in the molecular weight range of 48,000-50,000. In this study we used a strategy for the identification and isolation of this transport protein based on the observation that Na(+)-dependent transport activity is abruptly expressed in fetal rat liver just before birth [Suchy et al. Am. J. Physiol. 251 (Gastrointest. Liver Physiol. 14): G665-G673, 1986]. Analysis of basolateral plasma membranes by SDS-PAGE revealed that a protein of apparent molecular weight 48,000 was absent from fetal rat liver on day 19 of gestation, barely detectable on day 20, and thereafter increased progressively with postnatal development. Monospecific, polyclonal antibodies raised against the 48-kDa protein but not preimmune antibodies significantly inhibited the initial rate of Na(+)-dependent taurocholate uptake by isolated rat hepatocytes. In contrast, Na(+)-independent taurocholate transport and uptake of another anion, 35SO4(2-), were not affected by antibody treatment. When an extract containing the total complement of basolateral proteins was incorporated into asolectin liposomes, Na+ gradient-dependent uptake of taurocholate was observed, including a 2- to 2.5-fold accumulation of substrate above its equilibrium concentration (overshoot). However, if the membrane extract was first selectively depleted of the 48-kDa protein by immunoprecipitation with the anti-48-kDa antibody before reconstitution, Na(+)-dependent stimulation of taurocholate transport was completely abolished. These studies indicate that an ontogenically regulated 48-kDa protein is a component of the basolateral Na(+)-dependent transport system for bile acids.

Effect of antiserum to a 99 kDa polypeptide on the uptake of taurocholic acid by rat ileal brush border membrane vesicles

A 99 kDa polypeptide in rat ileal brush border membrane (BBM), regarded as a component of the active bile acid transport system on account of photoaffinity labeling, has been purified by affinity chromatography and preparative gel electrophoresis and utilized as an immunogen for raising polyclonal antibody. Immune serum, but not preimmune serum, specifically recognized a single band of 99 kDa protein on immunoblots of ileal and renal BBM. In contrast, no reactivity was observed with proteins in jejunal BBM. This polyclonal antibody, compared with preimmune serum and anticytosolic bile acid binding protein (14 kDa) serum, significantly inhibited the Na + dependent uptake of [ 3H] taurocholate by BBM vesicles (ϱ <0.01). [ 14C] D-glucose uptake by BBM vesicles was not influenced by the immune serum (ϱ <0.01). Thus, these studies provide further support for the specific role of a 99 kDa protein in ileal BBM bile acid transport.