Influence of bile salts on hepatic mdr2 P-glycoprotein expression

Advan. Enzyme Regul., Vol. 36, pp. 351-363. 19% Copyright @ 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain OOhS-2571/96/$32.0+ .OO

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INFLUENCE OF BILE SALTS ON HEPATIC mdr2 P-GLYCOPROTEIN EXPRESSION CHARLES M. G. FRIJTERS, ROELOF OTTENHOFF, MICHEL J. A. VAN WIJLAND, CARIN VAN NIEUWKERK, ALBERT K. GROEN and RONALD P. J. OUDE ELFERINK Department of Gastrointestinal and Liver Diseases, Academic Medical Center, Amsterdam, The Netherlands

INTRODUCTION

The hepatocyte is a polarized epithelial cell with a basolateral (sinusoidal) domain which is in contact with blood plasma and an apical (canalicular) domain which is the site of primary bile formation. Transport of nutrients and metabolites from the blood to the hepatocyte occurs by distinct transport processes in the sinusoidal membrane. The canalicular membranes of neighbouring hepatocytes form an extracellular lumen (bile canaliculus) that anastomosesinto the bile ductules to transport bile from the hepatocytes to the common bile duct. Hepatocyte derived metabolites, which need to be excreted, are transported across the canalicular membrane by specific transporters. Besides the excretory function of the hepatocyte for waste products, there is also secretion of bile salts which assist in fat dispersion and absorption in the intestinal tract. In addition, the canalicular membrane provides a site for extensive lipid secretion into bile. This is critical for cholesterol homeostasis and efficient absorption of dietary lipids in the intestine, while it also mitigates the detergent action of the high bile salt concentrations in the biliary tree. Several of the proteins which are responsible for transport across the canalicular membrane have now been defined at the functional level and some are known at the DNA level as well. Kamimoto et al. (1) demonstrated that transport of amphipatic drugs like daunomycin into canalicular membrane vesicles may be mediated by mdrl P-glycoprotein. It was subsequently recognized that a whole class of primary active ATP-dependent transporters mediate the efficient extrusion of organic compounds from the hepatocyte into bile (2). Until then, P-glycoproteins were only studied with regard to the phenomenon of multidrug resistance 351

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(MDR). Tumor cells, resistant towards a variety of cytotoxic drugs, acquired this resistance by overexpression of P-glycoproteins which actively pump these compounds out of the cell so as to keep the intracellular concentration low and thereby to protect the cell against toxicity [reviewed in (3, 4)]. The mammalian P-glycoproteins are encoded by a small family of structurally related genes, which include two members in humans (MDRl and MDR3) and three members in mice (mdrla, mdrlb and mdd). Pgps show a very high degree of inter- and intraspecies homology (3); nevertheless there is a striking functional difference between the individual mdr genes. Transfection of the MDRl, mdrla or mdrlb in cell lines reveals that these genes can confer multidrug resistance, while MDR3 and mdd apparently cannot (reviewed in (5)). It was demonstrated that P-glycoproteins are also expressed in normal tissues including the hepatocanalicular membrane (6-8). It is therefore assumed that the biliary secretion of amphipatic drugs may, at least partly, be mediated by mdrla/b Pgps in the mouse and their homologues in the rat and humans. Of all tissues tested, the liver is the organ of highest mdd Pgp expression (9) and the protein is localized in the canalicular membrane of the hepatocyte (7, 8). In order to elucidate the physiological function, transgenic knockout mice were produced in which each of the mdr-genes were inactivated (10, 11). Using knockout mice in which the gene for mdrla P-glycoprotein was disrupted, it was shown that this protein indeed is involved in the extrusion of organic cations and neutral compounds out of certain tissues including the liver (11). Recently we demonstrated that mdr2 P-glycoprotein in the canalicular membrane of the mouse plays an important role in the excretion of phospholipid into bile (10, 12). Mice with a homozygous disruption of the mdr2 gene (mdd -/-) showed a lOO-fold reduction of phospholipid in bile and the cholesterol excretion rate was greatly decreased as well. Mice with a heterozygous gene disruption (mdr2 +/-) showed intermediate excretion levels of phospholipid while cholesterol excretion levels were normal. This defect in biliary lipid secretion was not observed in knockout mice for the m&la gene (see Table 1). These results strongly suggest that mdr2 Pgp is a rate-controlling step in the secretion of phospholipids and demonstrate that the products mdrlalb and the mdr2 genes have entirely different functions. We have fitted our observations concerning lipid secretion in control vs mdr2 knockout mice, in the model that is described below (Figure 1). Mdd Pgp, localized in the canalicular membrane of the hepatocyte, translocates phospholipids from the inner leaflet of this membrane to the outer leaflet. Subsequently, bile salts present in the canalicular lumen interact with the

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TABLE 1. BILE SECRETION IN CONTROL MICE WITH A HOMOZYGOUS DISRUPTION OF THE m&la OR m&2 GENE Mouse genotype Bile flow (~gmVmin/lOOg) Bile salt secretion (nmol/min/lOO g) Phospholipid secretion (nmoUmin/lOO g) Cholesterol secretion (nmol/min/lOO g)

(+/+I

mdrla (-i-)

mdr2 (-i-)

9.8ir1.5 309+88 26.824.5 2.7kO.7

7.3k1.4 359+139 30.0f9.0 2.520.4

16.9+3.3* 394+88 0.3+0.5* 0.08+0.07*

Bile flow and bile components were determined in bile samples that were obtained during the first 10 min directly after canulation of the gallbladder. Data represent means +S.D. of seven animals from the (+/+) and the mdr2 (-/-) strain and from three animals from the mdrla (-/-) strain. Significant differences were determined by the student’s t-test. *p
outer leaflet of the membrane which ultimately leads to the secretion of phospholipids into the bile. The mechanism of the latter step is unknown but may involve vesiculation of phospholipids from the outer leaflet of the membrane. The suggestion that lipids are secreted from the canalicular membrane in a vesicular form comes from the electron-microscopic observation of such lipid vesicles in hepatocyte canaliculi (13). Support for the function of mdd Pgp as a phospholipid translocator was provided recently by two separate studies. Ruetz and Gros (14) expressed mdr2 Pgp in secretory vesicles of a yeast set mutant and demonstrated ATPdependent translocation of a fluorescent phosphatidylcholine analogue between the leaflets of the membrane. A study by Smith et al. (15) provided evidence that MDR3 Pgp, which is the human homologue of murine mdd Pgp, is able to induce translocation of phosphatidylcholine across the plasma membrane of fibroblasts that overexpress this protein. The proposed, speculative model takes into account that both mdd P-glycoprotein and bile salts are major factors regulating the secretion of biliary phospholipid. Regulation of Pgp Expression in the Liver

The expression of Pgps in rat liver has been studied under a variety of experimental conditions. These studies have clearly demonstrated that the gene regulation of mdrla/lb Pgp is very different from that of mdr2 Pgp. The expression of the mdrlallb gene was shown to be induced during cholestasis (16), hepatocarcinogenesis (17, 18), regeneration (U-20) or treatment with xenobiotics like acetyl-aminofluorene (21) or carbon tetrachloride (20). Induction of the mdr2 gene was observed during regeneration but not during cholestasis or carcinogenesis. After treatment with Ccl,, expression of the mdr2 gene was also induced but was less

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canaliculus

FIG. 1. Hypothetical model for the function of mdr2 Pgp in bile salt-dependent phospholipid secretion. In the absence of mdr2 Pgp (knockout mouse), no phospholipid secretion is observed; apparently the outer leaflet of the canalicular membrane is resistant towards the detergent action of high concentrations of bile salts that are pumped into the canalicular lumen (left panel). If present, mdr2 Pgp translocates phosphatidylcholine from the inner leaflet of the canahcular membrane at the expense of ATP hydrolysis. This creates a phospholipid imbalance in the membrane which may lead to unstable microdomains in the canalicular outer leaflet, but in the absence of bile salts this does not lead to phosphoiipid secretion (middle panel). Bile salts present in the canalicular lumen may further destabilize these microdomains so that vesicular structures are formed in the outer leaflet of the membrane which eventually could pinch off. An intermediate state in these vesicle could be inverted micelles (right panel).

eanaliculus

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outspoken and occurred during a shorter time interval than the m&l genes. During culture of rat hepatocytes, there is a strong induction of mdrlb but not mdr2 expression (22). All these data strongly suggest that the mdrl and mdr2 genes are under separate reguiatory control. Because of the physiological relationship between mdr2 Pgp-mediated phosphoIipid secretion and bile salt secretion, we investigated whether bile salts influence the expression of the mdr2 gene. MATERIALS

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METHODS

Control mice and mice which were heterozygous (+/-) or homozygous i-i-) for mdr2 gene disruption, with the genetic background of the FVB strain, were bred in our own coiony and were used for experiments at 2-5 months old. Animals were anesthetized by intraperitoneal injection of 1 mg/kg Hypnorm and 10 mg/kg diazepam. The abdomen was opened and after distal ligation of the ductus choledochus, the gallbladder was canulated. Bile samples were collected and immediately frozen at -20°C. Bile flow was determined gravimetrically assuming a density of 1 g/ml for bile. Total phospholipid, cholesterol and bile salt concentration were determined as described previously (12). Mice were fed a control diet of commercially obtained purified diet containing 20% (w/w) casein (K4068.02, Hope Farms, Woerden, NL). The experimental diet consisted of purified diet supplemented with Na-cholate 0.1% w/w or ursodeoxychoiate 0.5% w/w (UDC). All diets were administered during a 21 day period. Food and water were distributed ad libitum. For isolation of total RNA, the livers were excised, immediately freeze clamped, and stored at -80°C until further use. The total RNA was isoiated by an acidic guanidinium isothiocyanate-phenoI-chloroform extraction procedure described by Chomczynski and Sacchi (23). 10 pg RNA per sample was loaded on a 0.8% agarose gel containing 7% formaldehyde. Standard ethidiumbromide staining was used to check the integrity of the isolated RNA and to ensure the use of equal amounts of the material in each lane. Northern blotting was performed as described previously by Sambrook et al. (24) using Hybond N+. Subsequently, the blots were hybridized with different randomly primed, 3zP labelled probes at 65°C in 5 M sodium phosphate buffer, pH 7.5, containing 7% (w/v) SDS and 1 mM EDTA. The following probes were used: a 0.3 kb mdr2 cDNA fragment obtained after PstI-EcoRI digestion of a construct of a HincII-NaeI digested fragment of mdr2 (positions 1875-2168) cloned into SmaI-HincII digested pGEM-3Zf(-) as described by Smit et al.; a 1.3 kb PstI fragment of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. Both probes were kindly provided by Dr A. H. Schinkel

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of the Dutch Cancer Institute (10). After hybridizing, the blots were washed three times with 0.2xSSC +O.l% SDS at 65°C. Radioactivity on the blots was detected by autoradiography on Biomax MR film followed by detection using a Molecular Dynamics phosphoimaging device. RESULTS

The Relation between Biliary

AND

DISCUSSION

Phospholipid

and Bile Salt Secretion

It has long been known that phospholipid secretion into bile is driven by bile salts present in the canalicular lumen. Several studies have described a strong relationship between bile salt secretion and phospholipid or cholesterol secretion (12,25, 26). Increasing bile salt output levels leads to

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bile salt output (nmol/min.lOOg) FIG. 2. Bile salt-dependent phospholipid secretion in mice with different levels of mdr2 Pgp expression. The bile duct of control mice (closed circles) and of mice with a homozygous (open squares) or heterozygous (open circles) disruption of the mdr2 gene was cannulated and bile was collected for 90 min in 10 min intervals. During this period of bile collection, the bile salt pool in the circulation is depleted which leads to a time-dependent decrease in bile salt secretion. The rates of bile salt and phospholipid output during the consecutive collection periods are plotted against each other and this relation gives the dependence of phospholipid secretion on bile salt secretion. In mdr2 (-/-) mice there is virtually no phospholipid secretion. In mdr2 (+I-) mice, the relation between phospholipid and bile salt secretion still exists but at a reduced level. These data show that mdr2 Pgpfunction is a rate controlling step in bile-salt-dependent phospholipid secretion.

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an increase in phospholipid and cholesterol secretion until a plateau phase is reached (27,28). This phenomenon is illustrated for the mouse in Figure 2. In control (+/+) mice, a clear hyperbolic relation is observed between bile salt and phospholipid secretion. In mdr2 (-/-) mice, no phospholipid secretion was observed at any bile salt secretion rate. In heterozygous (+I-) mice for mdr2 gene disruption there was also a hyperbolic relation between bile salt and phospholiid secretion, but at a reduced phospholipid secretion level. It is also generally accepted that the secretion of biliary phospholipids is positively related to the hydrophobicity of the secreted bile salt species (reviewed in (29)). Hydrophobic bile salts like taurocholate and taurodeoxycholate give rise to a higher phospholipid secretion than hydrophilic bile salts like tauroursodeoxycholate and muricholate. The Influence of Bile Salts on mdr2 Expression

Because of this intimate relationship between bile salt and phospholipid secretion, we investigated the influence of bile salt composition on mdr2 Pgp expression and phospholipid secretion capacity. To this end, normal mice were fed diets to which bile salts of different hydrophobicity were added. The endogenous murine bile salt pool consists mainly of the hydrophilic bile salt muricholate (88%), the remainder being taurocholate. In order to increase the proportion of hydrophobic bile salts in the circulation, mice were fed a purified diet to which 0.1% cholate was added, which is quantitatively converted into taurocholate in the liver. This led to an almost complete replacement of the bile salts by taurocholate (80%), which is more hydrophobic than the main endogenous bile salt, muricholate. Alternatively, mice were fed a purified diet to which 0.5% ursodeoxycholate was added; the hydrophobicity of this bile salt is comparable to muricholate. The bile salts in these mice consisted mainly of ursodeoxycholate and muricholate (87% and 7%. respectively). Feeding mice with cholate resulted in an increased slope of the relation between phospholipid and bile salt secretion (Figure 3A). On the other hand, ursodeoxycholate feeding failed to change this relation (Figure 3B). Two factors can play a role in this phenomenon; since it was shown that mdd Pgp is a rate controlling step in phospholipid secretion (see Figure 2), this might indicate that cholate feeding leads to an increase in the expression of the mdr2 gene. On the other hand, the increased phospholipid secretion will, at least partially, be caused by the fact that cholate feeding leads to an increased hydrophobicity of the secreted bile salts and as a consequence to increased phospholipid secretion. To evaluate the contribution of increased mdr2 gene expression, liver mRNA levels were assessedby Northern blotting with a mdr2 cDNA probe. Feeding with the

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bllo salt output (nmol/min.tOOg) FIG. 3. Bile-salt-dependent phospholipid secretion in mice on cholate and ursodeoxycholatesupplemented diets. Normal mice were fed a purified diet (open squares) to which either 0.1% cholate (closed squares, panel A) or 0.5% ursodeoxycholate (closed circles, panel B) was added. After three weeks on this diet, the bile duct was cannulated and bile-salt-dependent phospholipid secretion was determined as described in Figure 2. Panel A shows that cholate feeding leads to an increased phospholipid secretion capacity. Panel B shows that ursodeoxycholate feeding has no influence on the phospholipid secretion capacity.

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mdr2

GAPDH

FIG. 4. Induction of mdr2 mRNA level in mice fed with a cholate-suoulemented diet. Normal mice were fed either a control diet (lane 1) or a diet to which 6.1% cholate was added (lane 2). After 3 weeks, the liver was excised and processed immediately for mRNA extraction. Northern blotting of mRNA with an mdr2 probe revealed increased expression in the liver of the cholate-fed mouse. As an internal control. the blot was also probed for GAPDH.

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hydrophobic bile salt cholate resulted in an induction of mdd mRNA levels compared to mice on a control diet (Figure 4). The observed induction of mdd Pgp expression was already maximal within one day of the start of the dietary period and could not be observed after ursodeoxycholate feeding (not shown). These results suggest that the expression of the mdr2 gene is physiologically regulated by the type of bile salt present in the circulation, hydrophobic bile salts being more effective in inducing gene expression than their hydrophilic counterparts. The Function of Biliary

Lipid Secretion and its Regulation by Bile Salts

What is the physiological relevance of bile salts regulating mdd Pgp expression? As mentioned before, one of the important functions of biliary lipid secretion is the inactivation of the detergent action of bile salts in the biliary tree. This function is rigorously demonstrated by the liver pathology in mdd knockout mice. In the absence of biliary lipid secretion, these animals suffer from hepatocyte necrosis, ductular proliferation, portal inflammation and finally primary hepatocellular carcinoma (30). The progressive character of this pathology is determined by the hydrophobicity of the bile salt pool in these animals. When mdd (-/-) mice are fed a cholate-supplemented diet, there is a more rapid and more severe development of histological aberrations (31). These observations can provide a physiological explanation of the observed increase in mdd Pgp expression by hydrophobic bile salts. In the case of secretion of more hydrophobic bile salts into the canaliculus, more lipid will be needed to inactivate the lytic action of these bile salts. And because the detergent action of hydrophobic bile salts (e.g., taurocholate) is stronger than that of hydrophilic bile salts (e.g., tauroursodeoxycholate), this can explain the upregulation of mdr2 Pgp by cholate and the absence of upregulation by ursodeoxycholate. The Role of Bile Salts in Gene Regulation

Bile salts have been described before as potential regulators of gene expression; they are able to reduce gene transcription of keyenzymes in their own synthetic pathway, 7a-hydroxylase and sterol 27-hydroxylase (32-35). The regulatory capacity seems to be dependent on the hydrophobicity and structure of bile salts. Several studies describe involvement of a proximal 5’-flanking region of the rat cholesterol 701hydroxylase gene in the regulation of this gene by bile salts (36, 37). This bile-acid-responsive element was further examined by DNAse I footprinting. Chiang et al. (37) have proposed a model for the bilesalt-dependent regulation, in which bile salts bind to a bile salt receptor

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and displace a positive tram-acting factor from the proximal cholesterol 7a-hydroxylase gene. Stravitz et al. (38) have recently described that the repression of transcription of this gene is mediated through a proteinkinase-C-dependent mechanism. They suggestthat PKC or PKC-dependent transcription factors antagonize the activity of positive trans-acting factors residing in the 5’-flanking region of the cholesterol 7cu-hydroxylase gene. Our observation that a change in phospholipid output capacity is accompanied by an equal increase in mdr2 Pgp mRNA favors a direct effect of bile salts on transcription of the mdr2 gene. The regulation of the mdr2 gene could be achieved by a similar mechanism as that postulated for the expression of 7cu-hydroxylase, although the effects on this gene are reciprocal to those on the mdd P-glycoprotein expression. SUMMARY

Mdr2 P-glycoprotein is expressed in the canalicular membrane of the mouse hepatocyte and is responsible for phospholipid secretion into bile. It is our hypothesis that it functions as a flippase in the translocation of phosphatidylcholine from the inner leaflet to the outer leaflet of the canalicular membrane. We have investigated the influence of different types of bile salts on the expression levels of mdd Pgp. Feeding mice a cholate-supplemented diet results in an increased mdr2 mRNA level, and this is accompanied by an increased biliary phospholipid secretion capacity. Cholate is a more hydrophobic bile salt than the main endogenous bile salt, muricholate. The induction of mdr2 gene expression and concomitant increase in phospholipid secretion are in line with the function of biliary phospholipids to inactivate the detergent action of hydrophobic bile salts. REFERENCES 1. Y. KAMIMOTO, Z. GATMAITAN, J. HSU and I. M. ARIAS, The function of Gp170, the multidrug resistance gene product, in rat liver canalicular membrane vesicles, J. Biol. Chem. 264, 11693-11698(1989). 2. I. M. ARIAS, M. CHE, Z. GATMAITAN, C. LEVEILLE, T. NISHIDA and M. ST. PIERRE, The biology of the bile canaliculus, Hepufology 17, 31&k329(lYY3). 3. J. A. ENDICOTT and V. LING, The biochemistry of P-glycoprotein-mediated multidrug resistance, Ann. Rev. Biochem. 58, 137-171 (1989). 4. M. M. GOTTESMAN and I. PASTAN, Biochemistry of multidrug resistance mediated by the multidrug transporter, Ann. Rev. Biochem. 284, 278-284 (1993). 5. P. BORST, A. H. SCHINKEL, J. J. M. SMIT, E. WAGENAAR, L. VANDEEMTER, A. J. SMITH, E. W. H. M. EIJDEMS, F. BAAS and G. J. R. ZAMAN, Classical and novel forms of multidrug resistance and the physiological functions of P-glycoproteins in mammals, Pharmacol. Ther. 60, 289-299 (1993). 6. F. THIEBAUT, T. TSURUO, H. HAMADA, M. M. GOTTESMAN, I. PASTAN and M. C. WILLINGHAM, Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues, Proc. Nat1 Acad. Sci. U.S.A. 84, 77357738 (1987).

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7. E. BUSCHMAN, R. J. ARCECI, J. M. CROOP, M. X. CHE, I. M. ARIAS, D. E. HOUSMAN and P. GROS, mdr2 encodes P-glycoprotein expressed in the bile canalicular membrane as determined by isoform-specific antibodies, J. Viol. Chem. 267, 18093-18099 (1992). 8. I. J. M. SMIT, A. H. SCHINKEL, C. A. A. M. MOL, D. MAJOOR, W. J. MOOI, A. P. M. JONGSMA, C. R. LINCKE and P. BORST, Tissue distribution of the human MDR3 P-glycoprotein, Lab. Invesr. 71, 638-649 (1994). 9. J. E. CHIN, R. SOFFIR, K. E. NOONAN, K. CHOI and I. B. RONINSON, Structure and expression of the human MDR (P-glycoprotein) gene family, Mol. Cell. Biol. 9, 3808-3820 (1989). 10. J. J. M. SMIT, A. H. SCHINKEL, R. P. J. OUDE ELFERINK, A. K. GROEN, E. WAGENAAR, L. VAN DEEMTER, C. A. A. M. MOL, R. OTTENHOFF, N. M. T, VAN DER LUGT, M. A. VAN ROON, M. A. VAN DER VALK, G. J. A. OFFERHAUS, A. J. M. BERNS and P. BORST, Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease, Cell 75, 451-462 (1993). J. H. BEIJNEN, E. 11. A. H. SCHINKEL, J. J. M. SMIT, 0. VANTELLINGEN, WAGENAAR, L. VANDEEMTER, C. A. A. M. MOL, M. A. VANDERVALK, E. C. ROBANUSMAANDAG, H. P. J. TERIELE, A. J. M. BERNS and P. BORST, Disruption of the mouse mdrla P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs, Cell 77, 491-502 (1994). 12. R. P. J. OUDE ELFERINK, R. O~ENHOFF, M. J. A. VAN WIJLAND, J. J. M. SMIT, A. H. SCHINKEL and A. K. GROEN, Regulation of biliary lipid secretion by mdr2-P-glycoprotein in the mouse, J. Clin. lnvesf. 95, 31-38 (1995). 13. N. ULLOA, J. GARRIDO and F. NERVI, Ultracentrifugal isolation of vesicular carriers of biliary cholesterol in native human and rat bile, Hepatology 7, 235-244 (1987). 14. S. RUETZ and P. GROS, Functional expression of P-glycoproteins in secretory vesicles, J. Biof. Chem. 269, 12277-12284(1994). B. ROELOFSEN, K. 15. A. J. SMITH, J. L. P. M. ~MMERMANS-HEREIJGERS, W. A. WIRTZ, W. J. VAN BLIT-I-ERSWIJK, J. J. M. SMIT, A. H. SCHINKEL and P. BORST, The human MDR3 P-glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice, FEBS Len. 354, 263-266 (1994). 16. D. SCHRENK, T. W. GANT, K.-H. PREISEGGER, J. A. SILVERMAN, P. A. MARINO and S. S. THORGEIRSSON, Induction of multidrug resistance gene expression during cholestasis in rats and nonhuman primates, Heparofogy 17, 854860 (1993). R. P. EVARTS, R. K. BURT, P. NAGY and S. S. 17. H. NAKATSUKASA, THORGEIRSSON, Cellular pattern of multidrug-resistance gene expression during chemical hepatocarcinogenesis in the rat, Mol. Carcinogen. 6, 190-198 (1992). 18. S. S. THORGEIRSSON, B. E. HUBER, S. SORRELL, A. FOJO, I. PASTAN and M. M. GOTTESMAN, Expression of the multidrug-resistant gene in hepatocarcinogenesis and regenerating rat Iiver, Science 236, 1120-l 122 (1987). 19. L. D. TEETER, M. ESTES, J. Y. CHAN, H. ATASSI, S. SELL, F. F. BECKER and M. T. KUO, Activation of distinct multidrug-resistance (P-gIycoprotein) genes during rat liver regeneration and hepatocarcinogenesis, Mol. Carcinogen. 8, 67-73 (1993). 20. H. NAKATSUKASA, J. A. SILVERMAN, T. W. GANT, R. P. EVARTS and S. S. THORGEIRSSON, Expression of multidrug resistance genes in rat liver during regeneration and after carbon tetrachloride intoxication, Hepatology 18, 1202-1207 (1993). A. ORZECHOWSKI, J. A. 21. D. SCHRENK, T. W. GANT, A. MICHALKE, SILVERMAN. N. BATTULA and S. S. THORGEIRSSON. Metabolic activation of 2-acetylaminofluorene is required for induction of muitidrug resistance gene expression in rat liver cells, Curcinogerzesis15, 2541-2546 (1994). 22. C. H. LEE, G. BRADLEY, J. T. ZHANG and V. LING, Differential expression of

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