Many of the compounds taken up by the liver are organic anions that circulate tightly bound to protein carriers such as albumin. The fenestrated sinusoidal endothelium of the liver permits these compounds to have access to hepatocytes. Studies to characterize hepatic uptake of organic anions through kinetic analyses, suggested that it was carrier-mediated. Attempts to identify specific transporters by biochemical approaches were largely unsuccessful and were replaced by studies that utilized expression cloning. These studies led to identification of the organic anion transport proteins (oatps), a family of 12 transmembrane domain glycoproteins that have broad and often overlapping substrate specificities. The oatps mediate Na(+)-independent organic anion uptake. Other studies identified a seven transmembrane domain glycoprotein, Na(+)/taurocholate transporting protein (ntcp) as mediating Na(+)-dependent uptake of bile acids as well as other organic anions. Although mutations or deficiencies of specific members of the oatp family have been associated with transport abnormalities, there have been no such reports for ntcp, and its physiologic role remains to be determined, although expression of ntcp in vitro recapitulates the characteristics of Na(+)-dependent bile acid transport that is seen in vivo. Both ntcp and oatps traffic between the cell surface and intracellular vesicular pools. These vesicles move through the cell on microtubules, using the microtubule based motors dynein and kinesins. Factors that regulate this motility are under study and may provide a unique mechanism that can alter the plasma membrane content of these transporters and consequently their accessibility to circulating ligands.

Obstructive jaundice occurs in patients suffering from cholelithiasis and from neoplasms affecting the pancreas and the common bile duct. The absorption, distribution and elimination of drugs are impaired during this pathology. Prolonged cholestasis may alter both liver and kidney function. Lactam antibiotics, diuretics, non-steroidal anti-inflammatory drugs, several antiviral drugs as well as endogenous compounds are classified as organic anions. The hepatic and renal organic anion transport pathways play a key role in the pharmacokinetics of these compounds. It has been demonstrated that acute extrahepatic cholestasis is associated with increased renal elimination of organic anions. The present work describes the molecular mechanisms involved in the regulation of the expression and function of the renal and hepatic organic anion transporters in extrahepatic cholestasis, such as multidrug resistance-associated protein 2, organic anion transporting polypeptide 1, organic anion transporter 3, bilitranslocase, bromosulfophthalein/bilirubin binding protein, organic anion transporter 1 and sodium dependent bile salt transporter. The modulation in the expression of renal organic anion transporters constitutes a compensatory mechanism to overcome the hepatic dysfunction in the elimination of organic anions.

Bilitranslocase is a carrier protein localized at the basolateral domain of the hepatocyte plasma membrane. It transports various organic anions, including bromosulfophthalein and anthocyanins. Functional studies in subcellular fractions enriched in plasma membrane revealed a high-affinity binding site for bilirubin, associated with bilitranslocase. The aim of this work was to test whether the liver uptake of bilirubin depends on the activity of bilitranslocase. To this purpose, an assay of bilirubin uptake into HepG2 cell cultures was set up. The transport assay medium contained bilirubin at a concentration of approximately 50 nm in the absence of albumin. To analyse the relative changes in bilirubin concentration in the medium throughout the uptake experiment, a highly sensitive thermal lens spectrometry method was used. The mechanism of bilirubin uptake into HepG2 cells was investigated by using inhibitors such as anti-sequence bilitranslocase antibodies, the protein-modifying reagent phenylmethanesulfonyl fluoride and diverse organic anions, including nicotinic acid, taurocholate and digoxin. To validate the assay further, both bromosulfophthalein and indocyanine green uptake in HepG2 cells was also characterized. The results obtained show that bilitranslocase is a carrier with specificity for both bilirubin and bromosulfophthalein, but not for indocyanine green.

BSP/Bilirubin binding protein (BBBP) is a protein located on the sinusoidal membrane of the liver that transport several organic anions. The aim of this investigation was to determine whether BBBP is present in the kidney and its role in p-aminohippurate transport (PAH). Anti-BBBP antibodies inhibited PAH uptake in brush border membrane vesicles (BBMV) and Na(+)-independent PAH uptake in basolateral membrane vesicles (BLMV). Western blot studies revealed positivity to antiBBBP antibodies in both BBMV and BLMV. So BBBP is also expressed in the kidneys and accounts, at least in part, for the renal tubular transport of PAH.

Enterohepatic recycling occurs by biliary excretion and intestinal reabsorption of a solute, sometimes with hepatic conjugation and intestinal deconjugation. Cycling is often associated with multiple peaks and a longer apparent half-life in a plasma concentration-time profile. Factors affecting biliary excretion include drug characteristics (chemical structure, polarity and molecular size), transport across sinusoidal plasma membrane and canniculae membranes, biotransformation and possible reabsorption from intrahepatic bile ductules. Intestinal reabsorption to complete the enterohepatic cycle may depend on hydrolysis of a drug conjugate by gut bacteria. Bioavailability is also affected by the extent of intestinal absorption, gut-wall P-glycoprotein efflux and gut-wall metabolism. Recently, there has been a considerable increase in our understanding of the role of transporters, of gene expression of intestinal and hepatic enzymes, and of hepatic zonation. Drugs, disease and genetics may result in induced or inhibited activity of transporters and metabolising enzymes. Reduced expression of one transporter, for example hepatic canalicular multidrug resistance-associated protein (MRP) 2, is often associated with enhanced expression of others, for example the usually quiescent basolateral efflux MRP3, to limit hepatic toxicity. In addition, physiologically relevant pharmacokinetic models, which describe enterohepatic recirculation in terms of its determinants (such as sporadic gall bladder emptying), have been developed. In general, enterohepatic recirculation may prolong the pharmacological effect of certain drugs and drug metabolites. Of particular importance is the potential amplifying effect of enterohepatic variability in defining differences in the bioavailability, apparent volume of distribution and clearance of a given compound. Genetic abnormalities, disease states, orally administered adsorbents and certain coadministered drugs all affect enterohepatic recycling.

The hypothesis that the uneven distribution of bilirubin in the organism, which occurs in hyperbilirubinemia, could reflect an uneven distribution of bilirubin-binding proteins was tested by searching for peptides containing the bilirubin-binding motif identified in bilitranslocase (Battiston et al., 1998). In the rat, positive proteins bands were found to be present only in the liver, gastric mucosa and central nervous system. The electrophoretic mobilities of the positive compounds in the liver and stomach were identical to that of purified bilitranslocase (38 kDa). In the brain, on the contrary, two peptides were found with molecular masses of 79 and 34 kDa, respectively. Their distribution pattern in the central nervous system was different for each of them.

In the primary structure of bilitranslocase, currently under study in our laboratory, an aminoacid motif was identified and found to be conserved in a number of alpha-phycocyanines, ancient biliproteins present in cyanobacteria. To test the possibility that such a motif could be at least part of the binding site for bilirubin, epitope-specific antibodies were raised. The target corresponds to the sequence 65-75 of bilitranslocase and covers the central portion of the motif identified. The antibodies were shown: 1) to inhibit the electrogenic BSP transport by plasmamembrane vesicles; 2) to react with purified bilitranslocase; and 3) to identify only one protein band with electrophoretic mobility identical to bilitranslocase in Western blots of solubilised plasmamembrane vesicles. The presence of either bilirubin or nicotinate during pre-incubation with the antibodies decreases concentration-wise the inhibition kinetics. From these experiments a dissociation constant of 2.2 +/- 0.3 and 11.3 +/- 1.3 nM for bilirubin-bilitranslocase and nicotinate-bilitranslocase complexes were calculated.

The renal handling of bilirubin in the rat was studied using an isolated kidney preparation by means of the determination of total pigment concentration decay in the perfusion medium and its renal clearance. Unconjugated bilirubin was incorporated in the perfusate at a concentration of about 4 microg/ml. In order to establish the potential role of secretion in renal handling of the pigment, experiments were also performed incorporating in the perfusate different doses of nicotinic acid (NA) (0.1 and 1.0 mM final concentration), which is considered an alternative substrate for the organic anion transport system, or probenecid (Prob) (0.1 and 1.0 mM final concentration), the classical inhibitor of organic anion transport process. The magnitude of pigment uptake from the perfusion medium, estimated by a first order exponential decay constant, was decreased in a dose-dependent way by NA (40 and 76% decrease for 0.1 and 1.0 mM of NA, respectively) and Prob (57 and 88% decrease for 0.1 and 1.0 mM of Prob, respectively). NA and Prob also induced a diminution in the ratio of pigment renal clearance to glomerular filtration rate (24 and 48% decrease for 0.1 and 1.0 mM of NA and 52 and 55% decrease for 0.1 and 1.0 mM of Prob). Based on these findings, it can be proposed that tubular secretion through the proximal cells contributes significantly to renal pigment depuration. In order to establish the possible contribution of cellular metabolism to the secretory process, a different set of experiments was conducted. The content of bilirubin mono and diconjugates (BMC and BDC) were determined in urine, in arterial and venous samples and in renal cortex. Studies performed using either an open or a closed circulating system, revealed that after conjugation in the renal cell, pigment derivatives can be secreted into both the tubule and the venous compartments. Total bilirubin concentration as well as the relative content of BMC and BDC in urine increased over time, representing the sum of both conjugates about 50% of the total pigment excreted by the end of experiments. Consequently, our results support the existence of a tubular transepithelial transport of bilirubin, playing the metabolism of the pigment an important role in this process.

Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB-HSA affinity constants (K'f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 microM, K'f (litre/mol) was inversely related to [HSA], irrespective of the [Bf]/[HSA] ratio. K'f was 2.066 x 10(6) + (3.258 x 10(8)/[HSA]). When 50 mM KC1 was isoosmotically substituted for sucrose, the K'f value was significantly lower {2.077 x 10(6) + (1.099 x 10(8)/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] < or = 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with K(m) values of 28 +/- 7 and 57 +/- 8 nM respectively (mean +/- S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112 +/- 12 versus 45 +/- 4 pmol of UCB. mg-1 of protein. 15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K'f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.

The sex difference in the hepatic uptake of tetrabromosulfonephthalein (TBS) was investigated in male and female rats in two different experimental models. In the intact animal, the initial plasma disappearance constant rate, the initial velocity of uptake, and the plasma clearance of TBS were significantly higher in females than in males. In sinusoidal liver plasma membrane vesicles, kinetic parameters of TBS uptake were investigated in both sexes. The Km was lower in females than in males (5.5 +/- 0.4 vs 17 +/- 4 microM, P < 0.05), whereas Vmax showed comparable values (544 +/- 15 vs 581 +/- 60 nmol TBS/min/mg protein, mean +/- SD, NS, females and males, respectively). Collectively, these data indicate that the sex difference in hepatic uptake of TBS is located at the sinusoidal liver plasma membrane and is due to a greater affinity of the electrogenic transport system(s) in females.

Muscle tissue (1.1 +/- 0.1 grams) was obtained from seven healthy individuals (3 males, 4 females) using an open incision approach before and after ingestion of either 75 grams of dextrose (N=5) or water (N=2). Purified sarcolemmal membranes from the muscle were prepared using a sucrose step gradient. A polyclonal antibody raised against the purified (99%) rat hepatocyte 40 KD membrane fatty acid binding protein (mFABP-L) was used to probe for this putative transporter in the muscle membranes using Western blot. A single band at the 40 KD MW band was identified which reacted antigenically with the protein purified from rat livers. These response of Berk's protein 60-75 minutes after dextrose ingestion (or water) was erratic and no specific trend could be identified. Our data demonstrate that the 40 KD mFABP-L originally isolated from rat liver is also present in human skeletal muscle membrane. This protein may be involved in transport of fatty acids across the membrane of skeletal muscle, however its physiological role in human fatty acid metabolism remains to be established.

Bile is an important excretory route for the elimination of amphiphilic organic anions, and hepatocytes are the primary secretory units of bile formation. The hepatocytic basolateral and canalicular membranes are equipped with various carrier proteins. Transport across the canalicular membrane represents a major concentrative step. Various ATP-dependent transporters have been identified, such as a multispecific organic anion transporter (canalicular multispecific organic ion transporter, cMOAT), a bile acid transporter and several P-glycoproteins. TR- rats, which lack cMOAT activity, have been valuable in defining the substrate specificity of cMOAT. A wide range of glucuronide-, glutathione- and sulfate-conjugates are transported by this system.

To determine whether phenobarbital affects hepatocellular bilirubin/sulfobromophthalein uptake mechanism, we administered it to male Sprague-Dawley rats, body weight 175 +/- 25 gm, at doses of 1 to 75 mg/kg body wt/day for 7 days. Control rats were given an equivalent volume of physiological saline solution. On day 8, hepatocytes were isolated by means of collagenase perfusion, suspended in Hanks' solution without albumin and incubated with high specific activity (3 Ci/mmol) [35S]sulfobromophthalein, which was synthesized in our laboratory and purified by means of a new reverse-phase high-pressure liquid chromatography procedure. The initial uptake rate of sulfobromophthalein was determined at sulfobromophthalein concentrations of 1 to 50 mumol/L with a rapid filtration technique. The maximum uptake velocity and Michaelis constant for sulfobromophthalein uptake at each phenobarbital dose were determined by means of a computer analysis. In control studies, maximum uptake and Michaelis constant were 48.0 +/- 16.7 (mean +/- S.D.) pmol/50,000 cells/min and 22 +/- 4 mumol/L, respectively. Maximum uptake velocity increased linearly with the log of the phenobarbital dose (r = 0.98, p < 0.01), the increase achieving statistical significance at a dose of 3 mg/kg/day. Michaelis constant, however, was essentially unchanged at phenobarbital doses of 50 mg/kg/day or less. The maximal observed increase in maximum uptake velocity of sulfobromophthalein (to 619% of control values) was appreciably greater than the maximal increase in UDP-glucuronyltransferase activity (200% of control) or immunoreactive ligandin concentrations (260% of control) seen in earlier studies, suggesting a direct effect on the plasma membrane transport mechanism.

The effect of different bile acids (BA) on the hepatic uptake of sulfobromophthalein (BSP) was investigated in liver plasma membrane vesicles enriched in basolateral fraction. BSP uptake was measured either in the absence (electroneutral component) or in the presence of a membrane potential (electrogenic component) induced by the addition of valinomycin in the presence of an inwardly-directed potassium gradient. BSP uptake was also measured in the presence of different BA [cholate (C), taurocholate (TC), ursodeoxycholate (UDC) and tauroursodeoxycholate (TUDC)]. Electrogenic BSP uptake was not affected by BA. Conversely, the electroneutral portion of the BSP uptake was inhibited with an inhibition constant (Ki, microM) of 230 +/- 40 for C, 103 +/- 33 for TC, 99 +/- 34 for UDC and 120 +/- 39 for TUDC, respectively (means +/- SD, N = 4). The Dixon and Cornish-Bowden plot of the data revealed an uncompetitive type of inhibition for each BA. These data indicate that the electroneutral, but not the electrogenic, BSP transport system is modulated by BA.

Thyroid hormone (TH) metabolism is altered in cases of unconjugated hyperbilirubinemia. These effects might involve inhibition of TH uptake by their target cells. Astrocytes, which are in close contact with the membranes of brain capillaries, might be the first brain cells to come into contact with bilirubin. Cultured rat brain astrocytes were used as a model to study the effects of bilirubin and bilirubin analogues on TH uptake. The initial uptake of [125I]T3 and [125I]T4 was inhibited by unconjugated bilirubin, biliverdin, ditaurobilirubin and bilirubin glucuronides. The inhibition of T3 uptake by the bilirubin analogues was competitive. The Ki values were: unconjugated bilirubin (31 microM), biliverdin (48 microM), ditaurobilirubin (2.5 microM) and bilirubin glucuronides (1.2 microM). This last value is similar to the Km of T3 transport (0.4 microM), indicating that bilirubin glucuronides have a high affinity for the TH transport system. By contrast, the uptakes of [3H]tryptophan and ]3H]glutamine were not inhibited. These results suggest that the astrocyte plasma membrane bears specific bilirubin-interaction sites that are closely related to the TH transport system. However, uptake of [14C]bilirubin by cultured astrocytes was a non-saturable process. Binding of bilirubin to the astrocyte plasma membrane may inhibit the TH uptake and impair their metabolism and their action on the intracellular targets.

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.

Uptake of organic anions into isolated rat hepatocytes was studied to examine their ATP dependency. In the presence of rotenone (0.2 microM), the initial velocity of the uptake (Vo) of dibromosulfophthalein (DBSP; 10 microM), 1-anilino-8-naphthalenesulfonate (ANS; 10 microM) and benzylpenicillin (PCG; 0.02 microM) was reduced to 60-70% of the control value, while that of bromosulfophthalein (BSP; 10 microM), rose bengal (RB; 10 microM) and bromophenol blue (BPB; 10 microM) was not affected. Furthermore, we examined the inhibitory effect of rotenone on the uptake at equilibrium of non-metabolizable ligands (DBSP, BPB and RB). The uptake of these ligands reached equilibrium at 30 min with a cel-to-medium concentration ratio (C/M ratio) of 75, 37 and 126, respectively. The C/M ratio at equilibrium of DBSP was reduced by rotenone to approx. 60% of the control value, while that of BPB and RB was not reduced. Other metabolic inhibitors such as sodium azide (10 mM) and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; 10 microM) also reduced the Vo of DBSP and PCG, while the uptake of BSP and RB was not reduced by these inhibitors. These results indicate that organic anions can be classified into two groups according to whether they are taken up by hepatocytes in an ATP-dependent manner, i.e., via active transport or in an ATP-independent manner, i.e., via facilitated diffusion. DBSP, PCG and ANS belong to the former group, whereas BSP, BPB and RB belong to the latter.

The uptake of bilirubin diglucuronide (BDG) into isolated rat hepatocytes was investigated in order to characterize the mechanism by which bile pigments are transported by the liver. The BDG uptake by hepatocytes was saturable. The uptake was inhibited by bilirubin, sulfobromophthalein, and bilirubin monoglucuronide, but not by taurocholate. The uptake was not affected by replacement of sodium with other cations except for choline. Only when sodium was replaced with choline, was significant decrease in uptake observed. When chloride was replaced with nitrate, BDG uptake decreased, but it was not changed by replacement with sulfate. Metabolic inhibitors did not affect BDG uptake significantly. Thus bile pigments share a common sodium-independent and electrogenic potential-dependent transporter in liver cell membranes. A high concentration of albumin interferes with BDG uptake.

Previous studies in cultured rat hepatocytes revealed that initial uptake of sulfobromophthalein (BSP) was markedly reduced upon removal of Cl- from the medium. In the present study, unidirectional Cl- gradients were established in short-term cultured rat hepatocytes and their effect on BSP uptake was determined. These investigations revealed that BSP uptake requires external Cl- and is not stimulated by unidirectional Cl- gradients, suggesting that BSP transport is not coupled to Cl- transport. In contrast, BSP transport is stimulated by an inside-to-outside OH- gradient, consistent with OH- exchange or H+ cotransport. As the presence of Cl- is essential for but not directly coupled to BSP transport, binding of 35S-BSP to hepatocytes was determined at 4 degrees C. This revealed an approximately 10-fold higher affinity of cells for BSP in the presence as compared to the absence of Cl- (Ka = 3.2 +/- 0.8 vs. 0.42 +/- 0.09 microM-1; P less than 0.02). Affinity of BSP for albumin was Cl(-)-independent, and was approximately 10% of its affinity for cells in the presence of Cl-. These results indicate that extracellular Cl- modulates the affinity of BSP for its hepatocyte transporter.

The EHBR is a mutant rat strain with congenital conjugated hyperbilirubinemia bred from a Sprague-Dawley rat. Transport of conjugated bilirubin, indocyanine green, and tetrabromosulfophtalein from liver to bile is severely impaired in these rats. Serum bilirubin amounts to 6.0 +/- 0.05 mg/dl (n = 4) in adult rats, with 97% conjugates. The bile flow is reduced to about 65% of the control group, whereas total bile acid in 10-min bile samples is similar. Liver histology of 10 week-old rats revealed neither intracellular pigmentation nor architectural abnormalities.

The hepatic plasma membrane fatty acid-binding protein (h-FABPPM) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate with a Ka approximately 1.2-1.4 x 10(7) M-1. Sinusoidally enriched liver plasma membranes and purified h-FABPPM have GOT enzymatic activity; the relative specific activities (units/mg) of the membranes and purified protein suggest that h-FABPPM constitutes 1-2% of plasma membrane protein in the rat hepatocyte. Monospecific rabbit antiserum against h-FABPPM reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of [3H]oleate but not that of [35S]sulfobromophthalein or [14C]taurocholate. The inhibition of oleate uptake produced by anti-h-FABPPM can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABPPM and mGOT are closely related.

Carrier-mediated transport of drugs occurs in various tissues in the body and may largely affect the rate of distribution and elimination. Saturable translocation mechanisms allowing competitive interactions have been identified in the kidneys (tubular secretion), mucosal cells in the gut (intestinal absorption and secretion), choroid plexus (removal of drug from the cerebrospinal fluid), and liver (hepatobiliary excretion). Drugs with quaternary and tertiary amine groups represent the large category of organic cations that can be transported via such mechanisms. The hepatic and to a lesser extent the intestinal cation carrier systems preferentially recognize relatively large molecular weight amphipathic compounds. In the case of multivalent cationic drugs, efficient transport only occurs if large hydrophobic ring structures provide a sufficient lipophilicity-hydrophilicity balance within the drug molecule. At least two separate carrier systems for hepatic uptake of organic cations have been identified through kinetic and photoaffinity labeling studies. In addition absorptive endocytosis may play a role that along with proton-antiport systems and membrane potential driven transport may lead to intracellular sequestration in lysosomes and mitochondria. Concentration gradients of inorganic ions may represent the driving forces for hepatic uptake and biliary excretion of drugs. Recent studies that aim to the identification of potential membrane carrier proteins indicate multiple carriers for organic anions, cations, and uncharged compounds with molecular weights around 50,000 Da. They may represent a family of closely related proteins exhibiting overlapping substrate specificity or, alternatively, an aspecific transport system that mediates translocation of various forms of drugs coupled with inorganic ions. Consequently, extensive pharmacokinetic interactions can be anticipated at the level of uptake and secretion of drugs regardless of their charge.

Cellular influx kinetics of 4-50 microM bilirubin diglucuronide and sulfobromophthalein (BSP) by the human hepatoma cell line Hep G2 was examined at 37 degrees C. In confluent monolayer cultures, cellular influx of increasing concentrations of conjugated bilirubin and BSP revealed similar saturation kinetics with Km values of 9.9 and 12.1 microM, and Vmax 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 degrees 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.

Cellular uptake of the cholephilic organic anion sulphobromophthalein (BSP) by the human biliary epithelium carcinoma cell line Sk-Cha-1 was examined at 37 degrees C. In confluent monolayer cultures the cellular influx rate of increasing concentrations of [35S]BSP followed saturation kinetics with a Km value of 18 microM and a Vmax value of 243 pmol.min-1.mg protein-1. Uptake of [35S]BSP was competitively inhibited by the presence of bilirubin diglucuronide, but not by taurocholate or cholate. Furthermore, uptake was temperature dependent with maximal cellular influx rates at 37 degrees C.

The binding of Rose bengal, a model organic anion, to sinusoidal and bile canalicular membrane fractions isolated from rat liver was compared. The fluorescence change of Rose bengal after being bound to liver plasma membranes was utilized for measuring the binding. The dissociation constants (Kd = 0.1-0.12 microM) and the binding capacities (n = 11-15 nmol/mg protein) for Rose bengal are comparable between the two membrane fractions, although the n value for sinusoidal membrane is somewhat larger than that for bile canalicular membrane. The Rose bengal binding to both membrane fractions was inhibited by various organic anions at relatively low concentrations, i.e., the half-inhibition concentrations (IC50) for Indocyanine green, sulfobromophthalein, Bromophenol blue and 1-anilino-8-naphthalene sulfonate were 0.1, 100, 1.5-2.5 and 100 microM, respectively, while taurocholate did not inhibit the Rose bengal binding to either membrane fraction at these low concentration ranges. The type of inhibition of sulfobromophthalein and Indocyanine green for Rose bengal binding is different between the two membrane domains. That is, in sinusoidal and bile canalicular membrane fractions, these organic anions exhibit mixed-type and competitive-type inhibition, respectively. It was suggested that the fluorescence method using Rose bengal may provide a simple method for detecting the specific organic anion binding protein(s) in the liver plasma membrane.

When freshly isolated well-stirred single cell suspensions of rat hepatocytes were incubated with 5-600 microM [3H]oleate or [35S]sulfobromophthalein (BSP) in the presence of 150 microM bovine serum albumin (BSA), uptake of both ligands increased as a linear function of the total ligand concentration in the medium. By contrast, when the same ligand concentrations were incubated as 1:1 complexes with BSA, apparent saturation of ligand uptake was observed. Analogous results were obtained in incubations employing beta-lactoglobulin instead of BSA. In none of these studies did ligand uptake velocity correlate in simple fashion with the concentration of unbound ligand in the incubation medium. These studies establish that the basis for the kinetic observations termed the 'albumin receptor phenomenon' does not require an intact hepatic lobular architecture or space of Disse, and is not specific for albumin.

Quantitation of initial uptake of the cholescintigraphy agents, 99mTc-Lidofenin, 99mTc-Disofenin, 99mTc-Mebrofenin, and 99mTc-Arclofenin, in short-term cultured rat hepatocytes revealed a marked reduction at 4 degrees C as compared with 37 degrees C. Depletion of adenosine triphosphate by preincubation of cells in sodium azide and 2-deoxyglucose reduced initial uptake of 99mTc-Disofenin at 37 degrees C by 50% (p less than 0.05), suggesting an energy-dependent mechanism. At 37 degrees C, 99mTc-Mebrofenin and 99mTc-Disofenin had the greatest rate of uptake. 99mTc-Disofenin and 99mTc-Lidofenin uptake was inhibited by 20 microM sulfobromophthalein, bilirubin, taurocholate, deoxycholate, chenodeoxycholate, and cholate, suggesting a common anionic transport mechanism. Uptake of 99mTc-Disofenin was unaffected by removal of NaCl from medium, suggesting that its transport did not proceed by the primary high-affinity uptake pathways associated with sulfobromophthalein and taurocholate, which require Cl- and Na+, respectively. 99mTc-Mebrofenin uptake was inhibited only modestly by taurocholate, deoxycholate, and bilirubin (p less than 0.05) and 99mTc-Arclofenin uptake was not inhibited by the organic anions studied. These results suggest that 99mTc-Mebrofenin and 99mTc-Arclofenin might be advantageous for cholescintigraphy in severely jaundiced patients. The relatively simple in vitro methodology described in this study may be useful in the design and screening of potential new agents before proceeding to animal studies or clinical trials.

A portion of the hepatocellular uptake of nonesterified long-chain fatty acids is mediated by a specific 40-kDa plasma membrane fatty acid binding protein, which has also been isolated from the gut. To investigate whether a similar transport process exists in other tissues with high transmembrane fatty acid fluxes, initial rates (Vo) of [3H]oleate uptake into isolated rat adipocytes were studied as a function of the concentration of unbound [3H]oleate in the medium. Vo reached a maximum as the concentration of unbound oleate was increased (Km = 0.30 +/- 0.03 microM; Vmax = 2470 +/- 90 pmol/min per 5 X 10(4) adipocytes) and was significantly inhibited both by phloretin and by prior incubation of the cells with Pronase. A rabbit antibody to the rat liver plasma membrane fatty acid binding protein inhibited adipocyte fatty acid uptake by up to 63% in dose-dependent fashion. Inhibition was noncompetitive; at an immunoglobulin concentration of 250 micrograms/ml Vmax was reduced from 2480 +/- 160 to 1870 +/- 80 pmol/min per 5 X 10(4) adipocytes, with no change in Km. A basic (pI approximately equal to 9.1) 40-kDa adipocyte plasma membrane fatty acid binding protein, isolated from crude adipocyte plasma membrane fractions, reacted strongly in both agar gel diffusion and electrophoretic blots with the antibody raised against the corresponding hepatic plasma membrane protein. These data indicate that the uptake of oleate by rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut.

The kinetics of bromo[35S]sulfophthalein (35S-BSP) binding by and uptake across the hepatocyte sinusoidal membrane were investigated using isolated rat liver sinusoidal membrane vesicles containing K+ as the principal internal inorganic cation. Uptake of 35S-BSP into vesicles was found to be temperature dependent, with maximum uptake between 35 and 40 degrees C; only binding occurred at or below 15 degrees C. Uptake at 37 degrees C was saturable and resolvable by Eadee-Hofstee analysis into two components: one with high affinity (Km = 53.1 microM) but low capacity, and the second of low affinity (Km = 1150 microM) but high capacity. By pre- or post-incubation, respectively, with unlabelled BSP, trans-stimulation and counter transport of 35S-BSP could also be demonstrated in these vesicles. Uptake was inhibited competitively using 5 microM Rose bengal and 10 microM indocyanine green, and non-competitively using 10 microM DIDS. Taurocholate did not inhibit uptake, and actually enhanced transport at concentrations greater than or equal to 250 microM. Imposition of inwardly directed inorganic ion gradients resulted in the enhancement of 35S-BSP transport when chloride ions were part of this gradient, irrespective of the cation employed whereas there was no apparent cation effect. However, substitution of 10 mM Na+ for 10 mM K+ as the internal cation resulted in a significant increase in uptake in the presence of external K+ as compared to Na+ gradients. This effect was not observed when 10 mM Tris+ was employed as the internal cation. The kinetics of 35S-BSP uptake by isolated sinusoidal membrane vesicles are indicative of facilitated transport. While the observed inorganic ion effects suggest a possible electrogenic component, the driving forces for hepatic BSP uptake remain uncertain. Isolated sinusoidal membrane vesicles provide a useful technique for studying hepatic uptake processes independent of circulatory or subsequent cellular phenomena.