Archives of
Toxicology
Arch Toxicol (1987) 60:37-42
9 Springer-Verlag 1987
Review
Drug transport in intestine, liver and kidney* Michael Schwenk Institut fiir Toxikologie der Universit~it, Wilhelmstral3e 56, D-7400 Tiibingen, Federal Republic of Germany
Abstract. Drug transport in intestine, liver and kidney is similar, because in each case transport occurs across a barrier of epithelial cells. However, the physiological conditions differ in each organ: intestinal drug absorption is largely influenced by physicochemical conditions in the intestinal lumen; actual transport across the epithelial barrier occurs mainly by diffusion; carrier-mediated transport plays a subordinate role. In contrast, hepatic uptake is mediated by specific carriers, which transport a wide variety of drugs into the liver cell and then release them either into bile, or back into the portal blood. It is unclear how many carrier systems are involved, how they are organized in the liver cell membrane, and to what extent their substrate specificities overlap. Renal secretion and reabsorption of drugs is mediated by highly active carrier systems for cations and anions. Their cooperative action results in either active reabsorption or active secretion of drugs. Key words: Intestine - L i v e r chanisms
Kidney -
Carrier me-
Introduction Intestine, liver and kidney are the major organs controlling the movement of drugs (non-volatile) in the organism. All three aim at protecting the organism from the intrusion of xenobiotics, either by preventing their absorption, or by detoxicating and eliminating them efficiently. For this purpose these organs are equipped with drug metabolizing enzymes on the one hand, and with carriers on the other hand, which eliminate the newly formed metabolites. Both systems supplement each other. If drugs were not absorbed, one would not require drug-metabolizing systems and if metabolites were not eliminated by specific transport systems, this metabolism would not be sufficient for rapid detoxification. While drug metabolism with its important practical implications is presently the center of interest, drug transport has attracted less interest. Consequently, molecular events of drug metabolism are better understood than those of drug transport. Drug transport in intestine, liver and kidney consists of movement of drugs across a barrier of epithelial cells. In * Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday Offprint requests to: M. Schwenk
each instance, drugs can move either from the blood into the cells or vice versa (Fig. l). This movement can occur by simple diffusion, or by specific processes. All permeation steps will be termed "transport" independent of their mechanisms. Despite the similiarity between the organs, the actual transport situation differs considerably. This presentation will describe some major characteristics of transscellular drug transport in the three organs. The interested reader will find extensive literature in the following reviews and books. Intestinal transport: Houston and Wood (1980); Csfiky (1984); Hoensch and Schwenk (1984). Hepatic transport: Meijer et al. (1983); Klaassen and Watkins (1984); Gebhardt 0986). Renal transport: Weiner (1971); Greger et al. (1981); Ross and Holohan (1983).
Methods of investigating drug transport Assessment of transport mechanisms is usually more complicated than resolution of conventional biochemical reactions, especially if drug are considered. This has several reasons. First, drugs have the disadvantage over most biochemicals that they are amphiphilic or lipophilic, thus tending to stick unspecifically to cellular membranes. Second, drugs are often transported via more than one route (e.g., diffusion plus carrier mediation), and transport usually involves two membrane permeations steps (uptake and release). Consequently, experimental results are usually composed of several overlapping reactions. The situation is further complicated in the case of active transport, where cellular energy supply and transmembraneous cation distribution are additionally involved. Because of these difficulties, drug transport mechanisms can only be resolved by application of a hierarchy of complementary methods. Whole animal studies are suitable to determine the general pharmacokinetics. However, quantitative data on the contribution of transport organs requires application of the isolated organs (either in situ or in vitro). These are also the tools of choice for elucidating whether transport occurs via the paracellular route or the transcellular route. For studies of carrier-mediated transport several additional methods are available. Isolated cells are a powerful tool for assessing intial rates of uptake or secretion. They allow study of the energetic coupling of transport (ATP or transmembrane gradients), and provide a convenient tool to disclose interdependencies between transport and biotransformation. They have, however, the disadvantage that
38 vitro. Carrier proteins can be labelled in the intact membrane by photoaffinity labeling methods with chemically reactive derivatives. The covalently labelled membrane components are then separated and characterized by modern analytical methods. appearanceof
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Mechanisms of intestinal transport
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Fig. 1. Schematic drawing of drug transport in intestine, liver and kidney the originally polar cells are entirely exposed to the substrate. It therefore remains obscure on which side (basal or luminal) of the polar cell the investigated transport system is located. To resolve this problem isolated membrane vesicles are required, which can be separately prepared f r o m the two sides of the polar cells. They also allow study of the energetics of drug transport and its relationship to cation movements. Despite thousands of studies on intestinal, hepatic and renal drug transport, it is unknown how many drug carriers exist in these organs, or how carriers for different drugs interact with each other. It appears that this question cannot be resolved with the described methods. An important future aim is therefore the isolation of the involved carriers and their further study in reconstituted systems in
The small intestine is the major route of entry of drugs into the organism. Figure 2 shows schematically the architecture of the intestinal wall. A single layer of epithelial cells forms the barrier between outside (lumen) and inside (blood capillaries). Cells are connected with each other by tight junctions. The epithelial cells control the entry of compounds. Their surface is decorated with short membrane protrusions (brush border), which contain digestive enzymes and carriers for uptake of nutrients. The basal part of these cells is embedded in the submucosa, which is rich in blood capillaries, lymphatic vessels, nerves and muscle fibers.
Five ways of drug absorption In principle drugs can surmount the mucosal barrier by five different routes and mechanisms (Fig. 3). The first mechanism is the paracellular transport through the tight junctions between cells. These junctions consist of several circular bands of proteinrich material beneath the brush border. These bands are not completely tight, but rather form a molecular sieve. This is permeable to water and small electrolytes. It is believed that even molecules with a considerable molecular weight, such as inulin, are absorbed to some extent via the paracellular pathway. Since a variety of drugs exhibit the same absorption kinetics as
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39 inulin, they might share the route via the paracellular pathway. Heavy metals may also pass through tight junctions, as visualized with lanthanum in electron microscopy studies. A major motive force for paracellular uptake is the solvent drag: most of the water, contained in the digestive juices (about 6 1 daily) is reabsorbed via the paracellular pathway. Solutes in this juice may be pulled through the tight junctions together with the water. The special case of paracellular uptake of solid particles is called persorption. These particles may pass through the loose junctions between the dying enterocytes of the villus tips. A second, rather unspecific route is that of transcellular diffusion. It is the route of absorption of small molucules and lipophilic molecules. Small xenobiotics, such as ethanol or nitrosodimethylamine are very rapidly absorbed from the intestine into the portal blood. Presumably they diffuse through the brush border membrane, travel through the aqueous phase of the cell and leave it at the basal membrane, via gaps in the protein-lipid interphases of the membrane. Highly lipophilic compounds like benzopyrene or D D T might also be absorbed by the transcellular route, though in a different way. They probably get integrated into the brush border membrane and move laterally along the plasma membrane towards the basal membrane. While paracellular transport and transcellular diffusion are completely unspecific events, the following mechanisms involve specific functions of the epithelial cells. It is believed that several heavy metals, such as mercury or cadmium and several organic drugs such as carbenoxolone are transported by facilitated diffusion. This mechanism requires the presence of two carriers in the epithelial cell: one for uptake at the brush border membrane, and a second for release at the basal membrane. Net movement occurs down the concentration gradient of the drug. Still more elaborate is the mechanism of carrier-mediated active transport. The cell provides the energy required for the concentrative uptake. In primary active transport systems, carriers get their energy by cleavage of ATP. Only a few carrier systems work this way (for example, the Napump). Most others work by a secondary active mechanism: the carrier utilizes the transmembraneous Na +-gradient and/or electrical potential as energy source for uphill transport of its substrate into the cell. The substrate then leaves the cell at the opposite pole by facilitated diffusion. Sugars, amino acids, nucleotides and many water-soluble vitamins are absorbed in this way. Some xenobiotics with structural similarity to nutrients share this way; examples are antibiotics with a peptide-like structure and 5-fluorouracil. Finally, absorption may occur by endocytosis, where the substrate gets engulfed by membrane protrusions which are internalized as vesicles. Though this process is slow, it may be of toxicological significance. There are actually three kinds of endocytosis (Dautry-Varsat and Lodish 1984): Phagocytosis is the internalization of solid matter, such as asbestos. Pinocytosis, is the internalization of small droplets; this is believed to be the mechanism of absorption of large molecules such as botulinus toxin. Receptor-mediated endocytosis is the internalization of molecules; it consists of three steps: substrate binding to a surface receptor, invagination of the receptor-substrate complex and delivery of the vesicle either to lysosomes (horse
radish peroxidase) or across the basal membrane into blood (absorption of maternal IgG in the newborn).
Physical and physiological factors affecting intestinal absorption A first precondition for effective absorption is the solubilization of the xenobiotic. Drug solubility may be restricted by specific interactions, such as complex formation between calcium and tetracycline or by unspecific events, such as the binding of T C D D to soil. Lipophilic xenobiotics can be solubilized by biliary micelles. In some cases this favors absorption; in others, however, it delays absorption, because micellar binding acts like a sink. Even if compounds are solubilized in the intestinal lumen, they are still separated from the brush border surface by three layers. The "unstirred layer" with a thickness of about 500 Ixm can only be crossed by diffusion and not by peristaltic propulsion. The "mucus layer" restricts diffusion and may have the properties of an ion exchange resin. The "acid microclimate layer" is a microlayer of mobile protons, opposing the fixed negative charges of the brush border surface. This layer is believed to favor the absorption of weak acids. Absorption is also affected by submucosal factors. Increased mesenteric blood flow favors absorption by several mechanisms: it improves oxygen supply, and increases the chemical concentration gradient across the mu,cosa by removing absorbed chemicals. Chemicals appear in the blood either freely dissolved (ethanol), or bound to albumin (most drugs), binding proteins (Fe), erythrocytes (Pb), and lipoproteins (chlorinated hydrocarbons); some appear in colloidal form (beryllium). Some lipophilic xenobiotics are integrated to some extent in chylomicrons (DDT) and appear in the lymphatic duct. A further important factor is the intestinal transit time. Drugs with rapid absorption are usually completely absorbed, independent of the mentioned modulating factors. Drugs with very slow absorption, however, react very sensitively on such modulation. It is well known from clinical observations that pharmaceutical drugs with slow absorption show a highly variable bioavailability between individuals. This relationship might also be applicable to toxic compounds.
Intestinal secretion The intestine is also a secretory organ and intestinal secretion appears to be major route for elimination of some heavy metals and some aromatic chlorinated hydrocarbons. Various mechanisms may lead to secretion: active secretion of cardiac glycosides, quaternary ammonium bases and some anionic dyes seem to involve carrier systems. Secretion of newly formed conjugates may also involve specific systems, though their nature is still unknown. A further possibility which has not been extensively studied would be release with secretory vesicles of goblet cells. Finally, xenobiotics may be released from the villus tips together with the dying epithelial cells; this may be important for highly peristent xenobiotics such as DDT.
Sites of intestinal transport Though most xenobiotics are believed to be absorbed in the small intestine, absorption may also occur in any other
40
Table 1. Transport systems in isolated hepatocytes 2 14 4 k~x~
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Inorganic cations Inorganic anions Endogenous intermediates Vitamins Hormones Amino acids Proteins Bile acid Xenobiotics
The table indicates different chemical groups, and the number of compounds in each group for which existence of specific hepatocellular uptake has been claimed (carriers or receptor-mediated endocytosis) and they transport drugs and conjugates into bile. None of these systems has been unequivocally characterized, and thus the mechanism of their function is still a mystery. Blood
Fig. 4. Schematic drawing of hepatocellular organization in the liver lobule; longitudinal (upper panel) and transversal (lower panel) sections
part of the gastrointestinal tract. The buccal mucosa avidly absorbs some compounds, and serves as the route of choice for nitroglycerine in an angina pectoris attack. The stomach preferentially absorbs weak acids, such as acetylsalicylic acid, but generally contributes less than 20% to total absorption; duodenum and jejunum are the major locations for absorption of nutrients and drugs because they posses a very large surface area, have comparatively leaky tight junctions and contain the biliary micelles required for solubilization of lipophilic compounds. The distal ileum is of physiological interest, because it contains highly specific carriers for uptake of cobalamin and bile acids. Colon and rectum readily absorb a variety of therapeutically used drugs.
Principles in hepatic transport After intestinal absorption drugs are carried with the portal blood to the liver, where they move from the periportal region of the lobules to the pericentral region. The specific architecture (fenestrated endothelium) of the hepatic sinusoids brings the drugs in close contact with the hepatocytes. These form a band of cells between blood and bile (Fig. 4) which is sealed by tight junctions. Drugs may be taken up from the blood into hepatocytes by different mechanisms. Some uncharged drugs such as nitrosodimethylamine, 1-naphthol or even PCBs appear to be taken up by diffusion. Others, such as iron (bound to transferrin) or drugs associated with lipoproteins may be taken up by receptor-mediated endocytosis. Inulin is taken up by fluid-phase endocytosis. Some heavy metals are taken up via cation channels, but a wide variety of drugs is transported by highly efficient drug-specific carrier systems in the sinusoidal membrane. These carriers will be considered in more detail. The hepatocellular drug-carrier systems can be functionally divided into three groups (Fig. 4, lower panel). They actively transport drugs into liver cells, they release newly formed conjugetes from the liver cell into the blood
Diversity of carriers for hepatic uptake The liver sinusoidal membrane is a highly dynamic structure, regulating the traffic of biomolecules and drugs. Table 1 lists compounds for which specific transport processes have been identified in isolated hepatocytes. Among them are about 20 xenobiotics. Many of the xenobiotics compete for transport with each other. Kinetic analysis allows distinction between four different types of carrier systems (Table 2) with partly overlapping specificities. Carrier 1 is identical with the active bile acid carrier but also transports a variety of drugs. In this secondary active transport system concentrative drug uptake is promoted by the transmembraneous Na § gradient (see below). The carrier covalently binds photoaffinity-labeled bile acids, labeled phalloidin (Wieland et al. 1984) and labeled DIDS (4,4'-diisothiocyanostilbene - 2,2'-disulfonic acid) (Ziegler et al. 1984). Carrier 2 transports a variety of organic anions, which contain strong as well as weak acide groups. These anions are considerably accumulated in hepatocytes. Since there is no evidence for cellular energy input, this accumulation appears to be predominantly caused by binding to intracellular macromolecules such as ligandin. A membrane protein has been isolated which may be identical with carrier 2: it binds bromosulphophtalein and bilirubin (Reichen and Berk 1979; Inagaki et al. 1985). Carrier 3 transports a variety of organic cations. The energetics are similar to those of carrier 1, but the
Table 2. Hypothetical carriers for hepatocellular drug uptake Carrier 1
Carrier 2
Carrier 3
Carrier 4
Bile acids Estronsulfate Estradiolglucuronide Iodipamide Phalloidin Antamanide
Bromsulphthalein Bilirubin ANS
Morphine Ouabain Nalorphine Procaine amide
Iopanoic acid Indocyanine green N-fluorescenyl-N-glycyl-thiourea
The table indicates four hypothetical carrier systems and their major substrates. For literature on the individual compounds cee Klaassen and Watkins (1984) and Gebhardt (1986)
41 transport systems interact only slightly with each other. Carrier 4 transports ouabain. It actively accumulates this compound in the liver cell. In contrast to carriers 1 and 3, it seems to serve as a primary active transport system (Schwenk et al. 1981). This hypothetical separation into four different carrier systems appears quite oversimplified if one considers that hundreds of drugs are secreted into bile. But it may provide a reasonable basis for further studies. We do not known whether these carriers for uptake are identical with those for the release into blood of newly formed conjugates.
Energetics of Na +-dependent uptake As mentioned, carrier 1 transports its substrates in an energy-dependent manner. However, the action of the carrier is not directly coupled to ATP (is not a primary active transport), but is coupled to the transmembraneous Na+-gradient. How can a Na+-gradient drive drug transport? A possible answer is given in the following hypothetical model which is designed according to models of the intestinal sugar transport mechanism (Restrepo and Kimmich 1985). The carrier protein is integrated into the phospholipid layer of the sinusoidal cell membrane. It binds Na § and this binding opens a hydrophobic binding site which attaches the drug with high affinity. The Na+-drug carrier complex undergoes a conformational change, whereby the binding sites become exposed to the cytoplasma. Since the intracellular Na+-concentration is low, Na + easily leaves its binding site. This induces a decrease in affinity for drug binding and the drug is released. The empty carrier may shuttle back to the extracellular surface. This model can explain how a Na+-gradient drives concentrative uptake. The concentrative force is further augmented if the carrier binds 2 Na+-ions (this may be the case with the bile acid carrier), for two reasons: first, the irreversibility of the reaction would be further increased (the probability that two Na+-ions bind to the carrier at the cytoplasmic surface is very small), and second, a positively charged carrier-complex would be pulled electrophoretically into the cell by the membrane potential.
the driving force seems to be provided by the membrane potential, which pushes the anions out of the cell (Meier et al. 1984). Alternatively, it has been assumed that bile acids and drugs might be secreted by exocytosis from Golgi vesicles (Simon et al. 1984). A further possible mechanism would be the association of amphiphilic drugs into the pericanalicular membranes. These might fuse with the canalicular membrane which then binds off its material into the canaliculus. The contractions of the canalicular membrane may support this process. The high bile/plasma concentration gradient (sometimes more than 1000) of many drugs is the result of various cell-biological events (active hepatocellular uptake, transcellular transport, active biliary secretion, ductular water reabsorption) and physicochemical interactions (intergration of drugs into pericanalicular membranes and mixed biliary micelles).
Localization of drug transport in the liver lobule The bile acid carriers seem to be rather homogenously distributed within the liver lobule. However, under physiological conditions the carriers work so efficiently that the bulk of bile acids is already taken up in the periportal region. Dibromsulfphthalein behaves similarly. Consequently, the drugs which are transported by these carriers get exposed perferentially to the drug metabolizing isoenzymes of this area. It is, however, possible that other drugs are preferentially taken up by the pericentral cells, in analogy to some biomolecules (glutamate).
Regulation of hepatic drug transport
Intracellular drug movement and canalicular secretion
Newborn rats transport ouabain and bile acids poorly. Postnatal transport is possibly induced by the increasing blood levels of substrates. Alterations of hepatic transport capacities occur in hepatomas (decreased bile acid transport), after phenobarbital induction (choleresis) and under a variety of hormones (cholestasis under estrogens). More knowledge is required on the half-life of the carriers, their diurnal regulation and their role in cholestasis. Regulation might either involve new synthesis (or loss) of carriers, or modification of carriers a n d / o r membranes by phosphorylation, methylation, reduction with GSH and other processes. These possibilities should be studied in more detail.
Drugs can move in various ways within liver ceils. Some are freely dissolved (ouabain), others are bound to proteins (bromsulfphthalein), associated with organelles (acridine orange) or converted by drug metabolizing enzymes. It is still an open question whether there are preformed routes from the sinusoidal to the canalicular membrane. Such routes could either consist of tubular systems which accumulate the drugs (glucuronides in ER?) or by vesicular systems such as endosomes (IgG) and Golgi (taurocholate). Microtubules and microfilaments seem to be involved in this directed transport, since their perturbation results in cholestasis. Future studies with time-lapse imaging methods may help uncover the secrets of intracellular movement. In any case, drugs get to the canalicular pool. Several mechanisms might lead to their secretion into bile. Studies with isolated hepatocytes and with canalicular membrane vesicle suggest that bile acid secretion is an active, carriermediated process. It is not dependent on a Na+-gradient;
In contrast to intestine and liver, the kidney has a filtration apparatus, which forces unbound drugs into the tubules. After filtration, weak acids (barbiturates) or weak bases (amphetamine) may be reabsorbed (in a pH-dependent manner) by diffusion. Strong acids and strong bases, however, behave differently. They are handled in the proximal tubule cells by specific transport processes (Fig. 5). Transport may occur in two directions: penicillin is almost exclusively secreted, while bile acids are almost exclusively reabsorbed. For many other drugs and conjugetes such as salicylic acid or uric acid, transport occurs in both directions. This is probably the cause for paradoxical clinical observations like the following: low doses of acetylsalicylic acid inhibit uric acid elimination, but high doses favor it. Analysis of proximal renal drug secretion led to the following model (Fig. 6): many anionic drugs are taken up
Renal drug transport
42 of lipophilic drugs is important for reabsorption; propulsion and pH o f the luminal fluid influence transport. As in liver, cells contain highly active but unspecific transport systems for anions and cations. Whether hepatic and renal drug carriers are genetically identical is a question for future research. In conclusion, transepithelial transport in intestine, liver and kidney is designed in such a way as to utilize nutrients at the highest possible efficacy and to reject xenobiotics from the organism as completely as possible. When rejection is not perfect, intoxication may occur.
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from the blood in a Na+-dependent active step (others in a Na+-independent manner). Their elimination at the brushborder site occurs by facilitated diffusion. The overall process leads to a 30-fold tubule/blood concentration difference for p-aminohippuric acid. Transport can be induced by substrates. Cations are taken up from the blood by facilitated diffusion; uptake is driven by the membrane potential. Their secretion into the lumen is coupled to antiport with protons.
Conclusions Drug transport in the intestine occurs mainly by diffusion. In addition, physicochemical conditions in the lumen and physiological events in the submucosa influence rates and directions of transport. Though some observations (secretion of conjugates and cardiac glycosides) suggest the presence of intestinal drug carriers, their contribution seems to be of minor importance. The liver is the organ where carrier-mediated drug transport predominates. Highly active but unspecific (towards substrates) carriers are integrated into the sinusoidal and probably into the canalicular membrane. These transport systems determine the fate of drugs and their metabolites in the body (blood or bile). Events in the kidney tubules are like a mixture between events in intestine and liver. As in the intestine, diffusion
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Csfiky TZ (1984) Intestinal absorption of xenobiotics. Handb Exp Pharmacol 70/II: 1-30 Dautry-Varsat A, Lodish HF (1984) How receptors bring proteins and particles into cells. Scientific American 250 (5): 48-54 Gebhardt R (1986use of isolated and cultured hepatocytes in studies on bile formation. In: Guillouzo A, Guguen-Guillouzo C (eds) Isolated and cultured hepatocytes. INSERM and John Libby, p 353 Greger R, Lanf L, Silbernagl S (eds) (1981) Renal transport of organic substances. Springer, Berlin, Heidelberg, New York Hoensch HP, Schwenk M (1984) Intestinal absorption and metabolism of xenobiotics in humans. In: Schiller CM (ed) Intestinal toxicology. Raven Press, New York, p 169 Houston JB, Wood SG (1980) Gastrointestinal absorption of drugs and other xenobiotics. In: Bridges JW, Chasseaud LF (eds) Progress in drug metabolism, Vol. 4. John Wiley, Chichester, p 57 Inagaki T, Stockert R J, Novikoff PM, Novikoff AB, Wolkoff AW (1985) Immunocytochemical localization of OABP in liver and heart. Hepatology 5:1018 Klaassen CD, Watkins III JB (1984) Mechanisms of bile formation, hepatic uptake, and biliary excretion. Pharmacol Rev 36:1-67 Meier PJ, Meier-Abt AS, Barrett C, Boyer JL (1984) Mechanisms of taurocholate transport in canalicular and basolateral rat liver plasma membrane vesicles. J Biol Chem 259: 10614-10622 Meijer DKF, Neef C, Groothuis GMM (1983) Carrier-mediated transport in the handling of drugs by the liver. In: Breimer DD, Speiser P (eds) Topics in pharmaceutical sciences. Elsevier, Amsterdam, p 167 Reichen J, Berk PD (1979) Isolation of an organic anion binding protein from rat liver plasma membrane fractions by affinity chromatography. Biochim Biophys Res Commun 91: 484-489 Restrepo D, Kimmich GA (1985) Kinetic analysis of mechanisms of intestinal Na+-dependent sugar transport. Am J Physiol 248: C498-C509 Ross CR, Holohan PD (1983) Transport of organic anions and cations in isolated renal plasma membranes. Ann Rev Pharmacol Toxicol 23:65-85 Schwenk M, Wiedmann T, Returner H (1981) Uptake, accumulation and release of ouabain by isolated rat hepatoeytes. Naunyn-Schmiedeberg's Arch Pharmacol 316:340-344 Simion FA, Fleischer B, Fleischer S (1984) Two distinct mechanisms for taurocholate uptake in subcellular fractions from rat liver. J Biol Chem 259:10814-10822 Weiner IM (1971) Excretion of drugs by the kidney. Handb Exp Pharmacol 28,1:328-353 Wieland T, Nassal M, Kramer W, Fricker G, Bickel U, Kurz G (1984) Identitiy of hepatic membrane transport systems for bile salts, phalloidin, and antamanide by photoaffinity labeling. Proc Natl Acad Sci 81 : 5232-5236 Ziegler K, Frimmer M, Fasold H (1984) Further characterization of membrane proteins involved in the transport of organic anions in hepatocytes. Biochem Biophys Acta 769:117-129