Cholephilic Organic Anions Research Articles

316 Cotransport of glutathione and bile acids via OATP8/1133. Potential role in the basolateral efflux of cholephilic organic anions from hepatocytes in cholestatic liver diseases

316 Cotransport of glutathione and bile acids via OATP8/1133. Potential role in the basolateral efflux of cholephilic organic anions from hepatocytes in cholestatic liver diseases

Excretion of Foetal Bilirubin by the Rat Placenta–Maternal Liver Tandem

Using plasma membrane vesicles from human trophoblast, carrier-mediated transport of unconjugated bilirubin (UCBR) has been reported. In the present work, using the in situ perfused rat placenta–maternal liver tandem, the relevance of this pathway in vivo was investigated. After single-pass perfusion of rat placenta through the umbilical artery with 0.25μmol [3H]-UCBR, ∼15 per cent of it was taken up by the placenta, detected in maternal serum (>96 per cent was unconjugated) and subsequently secreted into maternal bile (∼15 per cent of administered dose; >88 per cent was glucuronidated bilirubin). Co-administration through the umbilical artery of 0.25μmol [3H]-UCBR and 2.5μmol unlabelled UCBR, bromosulfophthalein, cholic acid or biliverdin IXα, reduced [3H]-UCBR placenta uptake, and the amount of radioactivity found in the maternal serum and bile. Co-administration into maternal jugular vein of 0.1μmol [3H]-UCBR—a dose 3-fold higher than that reaching the maternal compartment in placenta perfusion experiments—and 1.0μmol bromosulfophthalein, cholic acid or biliverdin IXα, resulted in no marked inhibition of the amount of radioactivity bile output. When antipyrine and [3H]-UCBR were continuously co-infused to the mother, similar antipyrine concentrations in maternal and foetal serum were reached in ∼15min, while progressive increase in [3H]-bilirubin concentrations in maternal serum above 70μm was accompanied by a very low transfer of this compound into foetal compartment where [3H]-bilirubin concentrations were always <10μm. These results suggest that the transfer of UCBR across the rat placenta occurs, without biotransformation, via a foetal-to-maternal mainly unidirectional pathway that can be cis-inhibited by UCBR and other cholephilic organic anions.

Expression of the hepatocyte canalicular multidrug resistance protein (MRP2) in primary biliary cirrhosis

The canalicular multidrug resistance protein 2 (MRP2; gene symbol: ABCC2) mediates ATP-dependent biliary excretion of organic anions such as bilirubin diglucuronide, glutathione conjugates and sulfated and glucuronidated bile salts. In chronic cholestatic liver diseases, the biliary excretion of cholephilic organic anions is impaired. While the underlying transport defects have been studied in rat models of cholestasis, little is known about the molecular basis of impaired organic anion excretion in human cholestatic liver disease. Our aim, therefore, was to analyze expression of MRP2 in patients with primary biliary cirrhosis (PBC), a chronic cholestatic liver disease characterized by progressive destruction of small intrahepatic bile ducts. Four patients with PBC stages III ( n=1) and IV ( n=3) were compared with three non-cholestatic patients with alcoholic liver disease, idiopathic liver cirrhosis and cirrhosis from chronic hepatitis C. Immunohistochemistry was performed on paraffin-embedded tissue slides using a monoclonal antibody to MRP2. MRP2 was detected at the canalicular hepatocyte membrane of all patients. In two PBC patients (stages III and IV, respectively), the degree of immunostaining was comparable with controls, whereas in two other PBC patients with stage IV disease immunostaining was decreased. We conclude that MRP2 expression decreases with progressive cholestasis in PBC.

Effect of phenobarbital on the expression of bile salt and organic anion transporters of rat liver

Background/Aims : The hepatic clearance of drugs and cholephilic organic anions is stimulated by phenobarbital (PB). Our aim was to analyze the effects of PB on the expression of hepatocellular bile salt and organic anion transporters. Methods : Male Sprague–Dawley rats were treated intraperitoneally with PB (80 mg/kg/d) or saline for 5 days. Transporter expression was quantified by northern and western blot analysis and initial uptake rates of bromosulphophthalein (BSP) and digoxin were measured in isolated hepatocytes. Results : Compared to control rats, PB treatment increased expression of the organic anion transporting polypeptide 2 (Oatp2; Slc21a5 ) more than 2-fold on the RNA ( P<0.05 ) and protein ( P<0.001 ) levels. Expression of Oatp1 ( Slc21a1 ), Oatp4 ( Slc21a6 ) and the Na +-taurocholate cotransporting polypeptide (Ntcp; Slc10a1 ) was unaltered. At the canalicular pole, expression of the bile salt export pump (Bsep; ABCB11 ) and of the multidrug resistance proteins 2 (Mrp2; ABCC2 ) and 6 (Mrp6; ABCC6 ) was not significantly changed. Whereas hepatocellular BSP uptake was unaffected by PB, digoxin uptake was stimulated 4-fold. Conclusions : The induction of digoxin uptake by PB correlates with Oatp2 expression. In contrast, the lack of increase of Oatp1 and Oatp4 expression is in accordance with unchanged BSP uptake. These data challenge the previously held view that PB induces hepatocellular BSP uptake systems.

Molecular structure influence in the recognition of phthaleins by the electrogenic organic anion carrier at the sinusoidal plasma membrane level in the liver.

The hepatocytic uptake of cholephilic organic anions occurs by carrier-mediated mechanisms. Electrogenic and electroneutral transport systems have been described. The aim of this study was to determine the dissociation constant (Kd) of the Electrogenic Carrier System(s) (ECS) for tetrabromosulfophthalein (BSP), dibromosulfophthalein (DBSP), tetrabromophthalein (TBP), tetrabromosulfonephthalein (TBS) and thymol blue (TB). Kd (uM) values for ECS-organic anion complexes were: ECS-BSP = 3.61 +/- 0.18; ECS-DBSP = 11.61 +/- 1.32; ECS-TBP = 0.51 +/- 0.08; ECS-TBS = 1.31 +/- 0.25; ECS-TB = 9.44 +/- 1.80. From these data, it is possible to conclude that molecular characteristics of the organic anions are important factors in determining the dissociation constant for the electrogenic hepatic carrier(s). In this sense, the addition of two sulphonic groups on the phenolic ring; the presence of a sulphonic on the benzenic ring and the absence of two or four bromines on the molecule confers a lower affinity for ECS.

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.

Functional characterization of the basolateral rat liver organic anion transporting polypeptide

To characterize the transport functions of a recently cloned basolateral organic anion transporting polypeptide of rat hepatocytes we performed further kinetic transport and substrate cis-inhibition studies in organic anion-transporting polypeptide-cRNA injected Xenopus laevis oocytes. The studies demonstrate saturable Na(+)-independent sulfobromophthalein (Michaelis-Menten constant, 1.5 mumol/L) and taurocholate (Michaelis-Menten constant, 50 mumol/L) uptake by organic anion-transporting polypeptide. Sulfobromophthalein uptake was inhibited by the following organic anions: 0.01 mmol/L bilirubin (43%), 0.1 mmol/L indocyanine green (81%), 0.1 mmol/L 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 52%) and 1 mmol/L probenecid (74%). Competitive inhibition was shown for indocyanine green (inhibition constant about 1.3 mumol/L). Sulfobromophthalein and taurocholate uptakes were also inhibited by cholate, chenodeoxycholate, deoxycholate and ursodeoxycholate, as well as their glycine and taurine conjugates. Organic anion-transporting polypeptide also mediated uptake of glycocholate, tauroursodeoxycholate and taurochenodeoxycholate. No cis-inhibition of sulfobromophthalein uptake was seen in the presence of ATP, para-aminohippuric acid, bumetanide, digitoxin, reduced glutathione, leukotriene C4, nicotinic acid, ouabain, oxalate, rifampicin, succinate or sulfate. Furthermore, radioactively labeled para-aminohippuric acid, alpha-ketoglutarate and reduced glutathione were not taken up by organic anion-transporting polypeptide in cRNA-injected frog oocytes. These data confirm that organic anion-transporting polypeptide represents a novel hepatocellular organic anion uptake system that can mediate Na(+)-independent transport of monovalent (e.g., bile acids) and divalent (e.g., sulfobromophthalein and indocyanine green) cholephilic organic anions.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of bumetanide transport in isolated skate hepatocytes.

The uptake of [3H]bumetanide was studied in isolated skate hepatocytes in an albumin-free elasmobranch Ringer solution and compared with the uptake of bile acids in the presence of other cholephilic organic anions. [3H]bumetanide uptake was energy dependent, temperature sensitive, and exhibited saturation kinetics. In contrast to taurocholate and cholate, which are transported only by Na(+)-independent mechanisms, removal of Na+ reduced the maximal uptake rate (Vmax) for bumetanide from 404 +/- 80 to 230 +/- 47 pmol.mg-1 x min-1 without a change in the apparent Michaelis constant (Km). The apparent Km for the Na(+)-dependent portion of bumetanide uptake was 58 +/- 24 microM, and Vmax was 151 +/- 38 pmol.min-1 x mg-1. Taurocholate (100 and 200 microM) inhibited Na(+)-independent bumetanide transport competitively but was a noncompetitive inhibitor for Na(+)-dependent bumetanide uptake. Furosemide (100 microM) and two bumetanide analogues, PF-3034 (500 microM) and PF-2203 (500 microM), preferentially inhibited the Na(+)-dependent bumetanide uptake system, whereas cholate (100 microM) and probenecid (100 microM) preferentially inhibited Na(+)-independent bumetanide transport. The sulfhydryl (SH) reagents N-ethylmaleimide, 2,2'-dithio-bis(5-nitropyridine), and p-chloromercuribenzenesulfonic acid (PCMBS) inhibited both bile acid and bumetanide uptake. Dithiothreitol (500 microM) completely reversed the PCMBS-induced inhibition of bumetanide uptake. These results indicate that bumetanide is transported into hepatocytes of the small skate, Raja erinacea, by both Na(+)-dependent and Na(+)-independent mechanisms; the latter is shared by bile acids and probably sulfobromophthalein and other organic anions. Their uptake requires free SH groups.

Difference in hepatic uptake of tetra- and Di-bromosulfophthalein in rat: Role of hydrophobicity, binding to plasma proteins and affinity for plasma membrane carrier protein

The relative role of hydrophobicity, binding to plasma proteins and affinity for one of the plasma membrane transport proteins in the hepatic uptake of 3,4,5,6-tetra- (BSP) and 3,6-di- (DBSP) bromosulfophthalein was investigated in the rat. In terms of physicochemical characteristics, the two molecules show different p K a , values and degrees of hydrophobicity, as determined from the n-octanol: water partition coefficient. In the intact animal, the plasma clearance and the plasma removal rate after a dose of 1.5 μmol/kg i.v. were significantly (P < 0.001) faster for BSP than DBSP, while no difference was found in the plasma distribution volume. The dissociation constant ( K d ) of the high affinity binding sites of plasma proteins also differed for the two anions, being significantly lower for BSP than DBSP (0.95 ± 0.02 vs 1.44 ± 0.14 μM, P < 0.001). [ 35S]BSP uptake by liver plasma membrane vesicles was saturable with an apparent K m of 5.20 ± 0.80 μM, and was competitively inhibited by DBSP ( K i 18.2 ± 1.2 μM) indicating a common uptake system. The K d value for binding of the organic anions to purified bilitranslocase, a plasma membrane protein involved in the electrogenic transport of pthaleins, was also significantly lower for BSP than DBSP (1.10 ± 0.12 vs 3.02 ± 0.27 μM, N = 3, P < 0.001), indicating a higher affinity of the former ligand for the carrier protein. No difference was observed in the capacity of the high affinity binding sites (32 ± 3 vs 33 ± 3 nmol/mg protein, BSP and DBSP, respectively). These data indicate that BSP and DBSP are two different cholephilic organic anions which share a common uptake mechanism, at least partly mediated by bilitranslocase. The greater affinity of BSP than DBSP for the carrier protein may account for the faster plasma disappearance rate of BSP observed in vivo, in spite of the higher plasma protein binding.

Bilitranslocase and sulfobromophthalein/bilirubin-binding protein are both involved in the hepatic uptake of organic anions.

The hepatic uptake of cholephilic organic anions is a carrier-mediated process. Three distinct proteins [bilitranslocase (BTL), sulfobromophthalein (BSP)/bilirubin-binding protein (BBBP), and organic anion-binding protein] have been isolated from the basolateral plasma-membrane domain of the hepatocyte. To investigate the relative role of the first two of them in accounting for the hepatic uptake of organic anions, we measured the initial rates of uptake of 35S-labeled BSP into rat liver plasma-membrane vesicles. Because transport by BTL is electrogenic but transport by BBBP is electroneutral, studies were done either with or without a positive-inside membrane potential produced by adding valinomycin in the presence of an inwardly directed K+ gradient (outside K+ > inside K+). Both electrogenic and electroneutral transport systems followed saturation kinetics. Electroneutral uptake showed an apparent Km of 20 +/- 3 microM (mean +/- SD) and a Vmax of 1.0 +/- 0.13 nmol.(mg of prot)-1.15 sec-1, whereas the electrogenic portion of BSP uptake exhibited a Km of 5.2 +/- 0.8 microM and a Vmax of 1.1 +/- 0.1 nmol.(mg of prot)-1.15 sec-1. In this case, an overshoot was observed 15 sec after valinomycin addition. Electroneutral BSP uptake was inhibited by incubation with anti-BBBP antibody, whereas anti-BTL antibody did not show any inhibitory effect. Conversely, the electrogenic uptake was inhibited by anti-BTL antibody at a BSP concentration of 5 microM; no inhibition was seen either at 20 microM BSP or upon addition of anti-BBBP antibody. From these data we conclude that the hepatic uptake of organic ions occurs via two immunologically distinct carrier proteins (BTL and BBBP) operating in parallel. BTL is a higher affinity electrogenic transporting system of organic ions, whereas BBBP is a lower affinity electroneutral transporter.

Interaction between cholephilic anions and bile acid transport across basal membrane of human trophoblast.

The sensitivity of radiolabeled bile acid (BA) binding and transport by basal plasma membrane (BPM) vesicles of human trophoblast to cholephilic organic anions (COAs) was studied by a rapid filtration technique. Glycocholate (GC) efflux from preloaded (15 microM GC) vesicles was investigated in the presence of 300 microM COAs at the trans-side of the membrane. Bilirubin (BR) diglucuronide and rose bengal induced a very strong transstimulating effect, whereas phalloidin and phenol red showed a negligible effect. This effect was from strong to moderate for indocyanine green > bromosulfophthalein (BSP) > or = fusidic acid > or = phenolphthalein > or = BR ditaurate > or = rifamycin SV > or = rifampicin. BSP-induced transstimulation was not additive to the "velocity effect" previously reported for bicarbonate. At the cis-side, BSP reduced the saturable component of taurocholate (TC) binding to BPM vesicles. BSP also induced a partial and mixed type of inhibition both in TC uptake [inhibitor constant (Ki) 227 microM] and efflux (Ki 209 microM). Two binding sites with overlapping specificity for BAs and other COAs are proposed in this carrier, the site for non-BA COA presumably corresponding to that for bicarbonate. In summary, the results indicate that several COAs can act as potential substrates for the BA carrier located at the BPM of human trophoblast. This stresses the "biliary-like" role of the placenta and suggests the possibility of developing new functional tests for this organ on the basis of fetal-maternal transfer of nontoxic cholephilic dyes.

A novel transcellular transport pathway for non-bile salt cholephilic organic anions

Non-bile salt cholephilic organic anions comprise a single class of nonhomologous ligands having a range of hydrophobicity. Hydrophobicity enhances the hepatic extraction of cholephiles as well as their partitioning into secreted biliary lipid particles. When hydrophobicity is correlated with patterns of biliary excretion for studying transcellular transport, however, the more hydrophobic probes are unsuitable. Specifically, with the isolated perfused rat liver technique, the excretory times for sulfobromophthalein and rose bengal were significantly longer compared with that for the much more hydrophilic analogue phenol red (PR), which showed only a single, nearly symmetrical excretory peak at 10 min. Colchicine affected the apparently well-defined PR pathway only at a saturation dose (10,000 times the tracer dose). In contrast, the effect of a different perturbant, monensin, was striking at a tracer dose of PR, but was less evident at a saturation dose. The combined administration of colchicine and monensin had no additive inhibitory effect on PR excretion at tracer doses. At a saturation dose of PR, where monensin is less inhibiting, however, a significant additive inhibitory effect was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition by colchicine of biliary secretion of diethylmaleate in the rat: Evidence for microtubule-dependent vesicular transport

It has been proposed that a microtubule-dependent transport of vesicles derived from the Golgi apparatus may play a role in biliary secretion of bile salts and other cholephilic anions. To test this hypothesis, we examined the influence of colchicine and vinblastine, two microtubule inhibitors, on diethylmaleate-induced bile flow and on the biliary secretion of diethylmaleate, an organic anion whose glutathione conjugates may be secreted into bile through the Golgi apparatus and Golgi-derived vesicles. Rats were pretreated with colchicine or vinblastine, and diethylmaleate was injected intraperitoneally at doses of 28 to 400 mumol/100 gm body wt. Basal bile flow was unaffected by colchicine or vinblastine. In contrast, diethylmaleate-induced bile flow and the secretion into bile of diethylmaleate conjugates (estimated by the cation-anion gap in bile) were significantly lower in colchicine-treated and vinblastine-treated animals than in controls. Diethylmaleate-induced bile flow was reduced in proportion to diethylmaleate conjugate secretion. A linear relationship was seen between bile flow and biliary output of diethylmaleate conjugates: this relationship was similar in colchicine-treated or vinblastine-treated animals and in controls. At electron microscopy, diethylmaleate had induced distension of the Golgi saccules of the hepatocytes. In conclusion, colchicine and vinblastine inhibited the secretion into bile of diethylmaleate conjugates and diethylmaleate-induced bile flow. These results support the view that microtubule-dependent transport of Golgi-derived vesicles is involved in the biliary secretion of diethylmaleate and, perhaps, other cholephilic organic anions.

Inhibition of glutathione-conjugate secretion from isolated hepatocytes by dipolar bile acids and other organic anions

The effect of a spectrum of organic compounds on the secretion of a model organic anion, dinitrophenylglutathione (GS-DNP), by hepatocytes was tested. Previous experiments have demonstrated that the secretion of GS-DNP from isolated rat hepatocytes is predominantly mediated by a canalicular transport system for this compound. Preincubation of isolated rat hepatocytes with the bile acids cholic acid (C), taurocholic acid (TC), tauroursodeoxycholic acid (TUDC) and glyco- or tauro-lithocholic acid (GLC or TLC) had no effect on the initial efflux rate of GS-DNP. In contrast, the 3-sulphates of GLC (SGLC) and TLC (STLC) did inhibit GS-DNP efflux; half-maximal inhibition with SGLC was reached with 10 microM. The 3-O-glucuronides of both cholate and lithocholate (GlucLC) were even more potent inhibitors of transport; 10 microM-GlucLC inhibited GS-DNP transport by 89%. Other cholephilic organic anions also inhibited GS-DNP secretion, albeit at higher concentrations; at 100 microM, bilirubin ditaurate, an analogue of bilirubin diglucuronide, inhibited transport by 48%. On the other hand, a number of cholephilic cationic and neutral compounds had no effect on GS-DNP efflux. The hepatobiliary secretion of oxidized glutathione (GSSG) was also investigated. In normal isolated perfused rat liver, extensive biliary secretion of GSSG was observed upon intracellular oxidation of reduced glutathione (GSH). GSSG was also actively secreted from isolated normal hepatocytes, and this secretion could be inhibited by 95% by incubation of the cells with 100 microM-SGLC. In contrast, biliary secretion was absent in the isolated perfused liver and in isolated hepatocytes from TR- mutant rats with a hereditary conjugated hyperbilirubinaemia. These results show that the canalicular efflux of GSSG and GS conjugates can be inhibited by a wide variety of polyvalent organic anions, but not by cations, neutral compounds and unianionic bile acids. This suggests that a multispecific organic-anion transporter is responsible for transport of these polyvalent anions, which is in close agreement with the fact that the biliary transport of all these compounds is defective in the mutant TR4 rat.

Hepatic extraction of organic anions in the rat depends on ligand hydrophobicity

Non-bile-salt cholephilic organic anions are efficiently taken up by the liver. Recent work from our group has suggested the possible importance of relative hydrophobicity among various organic anions in hepatic uptake. To further validate and clarify this, we studied hepatic extraction of five different cholephilic dyes using the isolated perfused rat liver in single-pass mode. Albumin binding affinities and capacities for each of the ligands were measured in vitro to permit evaluation of in vivo interactions for each of them over a spectrum of unbound ligand concentrations. As expected, a strong positive correlation was found between ligand hydrophobicity and the relative degree of albumin binding affinity and capacity. Using appropriate experimental conditions, we also found a strong positive correlation between hepatic extraction efficiency for a given ligand and both its hydrophobicity and its unbound concentration. These data indicate that where the unbound ligand concentration is significant, the greater the ligand hydrophobicity, the greater is its efficiency of hepatic extraction. We conclude that hepatic extraction efficiency for non-bile-salt cholephilic organic anions depends on a combination of ligand hydrophilic/hydrophobic balance and the availability of the unbound ligand for uptake.

Cellular uptake of conjugated bilirubin and sulfobromophthalein (BSP) by the human hepatoma cell line Hep G2 is mediated by a membrane BSP/bilirubin binding protein

Cellular influx kinetics of 4–50 μM bilirubin diglucuronide and sulfobromophthalein (BSP) by the human hepatoma cell line Hep G2 was examined at 37 °C. In confluent monolayer cultures, cellular influx of increasing concentrations of conjugated bilirubin and BSP revealed similar saturation kinetics with K m values of 9.9 and 12.1 μM, and V max values of 0.512 and 0.473 nmol·mg cell protein −1·min −1, respectively. Uptake of [ 3H]bilirubin diglucuronide was competitively inhibited by unlabeled BSP, and was temperature dependent with maximal cellular influx rates at 37 °C. When the confluent monolayer cultures were pretreated with a monospecific antibody to the rat liver BSP/bilirubin binding membrane protein, initial uptake rates of conjugated and unconjugated bilirubin as well as of BSP were significantly inhibited, whereas uptake of oleate was not affected. Furthermore, immunoblot analysis of the homogenate of Hep G2 cells with the same antibody revealed predominant reactivity with a 55 kDa protein. These data suggest that cellular uptake of bilirubin and related cholephilic organic anions by the human hepatoma cell line Hep G2 is mediated by a specific 55 kDa membrane BSP/bilirubin binding protein.

What's New in the Hepatocellular Uptake Mechanism of Bilirubin and Fatty Acids?

Although hepatocellular uptake of bilirubin and fatty acids represents a major function of the liver, little was known about the molecular mechanism of their translocation across the plasma membrane. This review provides evidence that these classes of albumin-bound organic anions enter hepatocytes via specific membrane associated carrier systems. After dissociation of the albumin-ligand complexes at the cell surface, bilirubin and related cholephilic organic anions bind to a 55 kDa membrane glycoprotein, while fatty acids bind to a 40 kDa membrane protein. Both proteins function as carrier of their respective ligand across the plasma membrane. Although the driving forces for the influx of bilirubin are still unknown, it could be demonstrated that the translocation of fatty acids across the plasma membrane is driven by an active, potential-sensitive, sodium-dependent transport system.

The role of albumin in the hepatic transport of bilirubin: studies in mutant analbuminemic rats.

Bilirubin and other cholephilic organic anions are bound to albumin in the circulation; their hepatic uptake involves a carrier-mediated process. To investigate the possible role of serum albumin in the transhepatic transport of a cholephilic ligand, plasma clearance of radioactive bilirubin and its biliary excretion as well as its interaction with plasma proteins were compared between normal and mutant analbuminemic rats (NAR). With a tracer amount of 3H-labeled bilirubin, its plasma clearance and biliary excretion were comparable in both animal groups. However, the plasma clearance of a loading dose of the ligand was significantly increased and its biliary recovery was low in NAR as compared with normal animals. In accord with these findings in vivo, gel permeation chromatographic analysis revealed that the bilirubin binding capacity of serum proteins was significantly lower in NAR than in control animals. When bilirubin was administered to NAR as a mixture with equimolar albumin, its plasma disappearance was considerably decreased and its biliary recovery was increased. Similar effects were observed when albumin was replaced by an equimolar amount of glutathione S-transferases (ligandins). These observations indicate that, although ligand-protein interaction in the circulation is important for directing bilirubin to the plasma membranes of the hepatocyte, this mechanism is not specific for albumin.

Transhepatic transport of taurocholic acid in normal and mutant analbuminemic rats

To elucidate a possible function of plasma albumin in vectorial transport of various cholephilic organic anions, such as bile acids, plasma clearance and transhepatic transport of radioactive taurocholate were studied in vivo in normal and mutant analbuminemic rats. Intravenous administration of taurocholate was followed by its rapid disappearance from the circulation in both animal groups. However, plasma clearance of taurocholate was significantly larger in analbuminemic (68.3 ml/min per kg of body weight) than in normal rats (29.8 ml/min per kg of body weight) at a dose of 8 μmol/kg of body weight. The increased plasma clearance in analbuminemic rats was accompanied by a more prompt biliary secretion of the ligand than occurred in normal animals; 79 and 42% of the injected dose was recovered in analbuminemic and normal rat bile, respectively, within 10 min after administration. Ultrafiltration analysis revealed that the binding of taurocholate to serum protein(s) was significantly lower in analbuminemic rats as compared with that in normal rat serum; 24 and 76% of taurocholate bound to protein fractions of analbuminemic and normal rat serum, respectively, at 0.5-mM ligand concentration. Binding of taurocholate to cytosolic proteins of normal and analbuminemic liver were similar; 23 and 28% of taurocholate bound to protein fractions from analbuminemic and normal rat, respectively, at 10 mg protein/ml and 20-μM ligand concentration. These results indicate that plasma albumin does not play a role in directing circulating taurocholate to the liver and that transhepatic transport of the bile acid increases with the increase in concentration of unbound ligand in the circulation.