Proc. Nati. Acad. Sci. USAVol. 88, pp. 6590-6594, August 1991Medical Sciences

Rat liver canalicular membrane vesicles contain an ATP-dependentbile acid transport systemToSHIROu NISHIDA, ZENAIDA GATMAITAN, MINGXIN CHE, AND IRWIN M. ARIAS*Department of Physiology, Tufts University School of Medicine, Boston, MA 02111

Communicated by G. Gilbert Ashwell, May 7, 1991 (received for review March 19, 1991)

ABSTRACT The secretion of bile by the liver is primarilydetermined by the ability of the hepatocyte to transport bileacids into the bile canaliculus. A carrier-mediated process forthe transport of taurocholate, the major bile acid in humansand rats, was previously demonstrated in canalicular mem-brane vesicles from rat liver. This process is driven by anoutside-positive membrane potential that is, however, insuffi-cient to explain the large bile acid concentration gradientbetween the hepatocyte and bile. In this study, we describe anATP-dependent transport system for taurocholate in inside-outcanalicular membrane vesicles from rat liver. The transportsystem is saturable, temperature-dependent, osmotically sen-sitive, specifically requires ATP, and does not function insinusoidal membrane vesicles and right side-out canalicularmembrane vesicles. Transport was inhibited by other bile acidsbut not by substrates for the previously demonstrated ATP-dependent canalicular transport systems for organic cations ornonbile acid organic anions. Defects in ATP-dependent cana-licular transport of bile acids may contribute to reduced bilesecretion (cholestasis) in various developmental, inheritable,and acquired disorders.

Taurocholate is the major bile acid in humans and rats. Itsuptake from plasma by hepatocytes utilizes a Na' cotrans-port system that requires Na',K+-ATPase for generation ofa Na' gradient (1, 2). Two pathways for intracellular bile acidtransport have been demonstrated. One involves microtu-bule-dependent vesicular transport from the Golgi apparatusto the bile canaliculus (3, 4). The other is the major physio-logic pathway and involves direct transport to the canalicu-lus, possibly by cytosolic binding proteins (5). The secretionof bile acids into the canaliculus is a major determinant ofbileflow. Although the contents ofthe bile canaliculus have neverbeen measured due to its inaccessibility, studies ofductal bilesuggest that there is a large bile acid concentration gradientbetween the hepatocyte and canalicular contents (6, 7).Previous studies using vesicles selectively derived from thecanalicular domain of hepatocyte plasma membrane revealedcarrier-mediated transport of taurocholate and other bileacids that is driven by an outside-positive membrane poten-tial (8, 9). However, the membrane potential is insufficient toexplain the concentration gradients of bile acids that arepostulated to occur across the canalicular membrane (6).We have described (10, 11) two distinct ATP-dependent

transport systems in the canalicular membrane. One systemutilizes P-glycoprotein, the product of the multidrug-resistance gene, and transports mainly hydrophobic cations,such as daunomycin (10). The carrier protein for the secondsystem has not been purified but its substrates are nonbileacid organic anions, such as bilirubin diglucuronide, oxidizedglutathione (GSSG), and glutathione S-conjugates (11-13).The latter transport system is defective in TR- mutant rats,which have a phenotype that resembles the defect in patients

with Dubin-Johnson syndrome (11, 14). While studying thesetransport processes, an ATP-dependent transport of bileacids in canalicular membrane vesicles (CMVs) was detectedand has been now characterized.

MATERIALS AND METHODSMaterials. [3H]Taurocholate, [3H]daunomycin, and [35S]_

sulfate were purchased from DuPont/New England Nuclear.Radiolabeled sulfobromophthalein ([35S]BSP) was synthe-sized as reported (15). The specific activity of [35S]BSP was2.0 Ci/mmol (1 Ci = 37 GBq). Daunomycin hydrochloride,BSP, adenosine 5'-[j,y-imido]triphosphate, adenosine 5'-[L,ymethylene]triphosphate, and goat anti-rabbit IgG wereobtained from Sigma. Sulfobromophthaleinyl glutathione(GSBSP) and dinitrophenyl glutathione (GSDNP) were syn-thesized nonenzymatically (16, 17). Rabbit polyclonal anti-body against the extracellular domain of -glutamyltrans-ferase (y-GT) was kindly provided by Masayasu Inoue (Ku-mamoto University Medical School, Kumamoto, Japan).Bilirubin diglucuronide, which was purified by HPLC (18),was kindly provided by N. Roy-Chowdhury and J. Roy-Chowdhury (Albert Einstein College of Medicine, NewYork). All other chemicals were of analytical grade.

Preparation of Plasma Membrane Vesicles. CMVs andsinusoidal membrane vesicles (SMVs) were separately pre-pared from male Sprague-Dawley rats weighing about 250 g.CMVs were isolated from rat liver using a nitrogen cavitationand calcium precipitation method (19). As compared withhomogenates, these preparations were enriched 62-fold and57-fold in 'y-GT and leucine aminopeptidase activities, re-spectively. Ouabain-inhibitable Na',K+-ATPase activitywas enriched 2-fold. SMVs were isolated from homogenatesusing differential and sucrose-Ficoll density gradient centrif-ugation (2). Ouabain-inhibitable Na',K+-ATPase activitywas enriched 24-fold in these preparations. CMVs and SMVswere stored in buffer A (10 mM Hepes'Tris, pH 7.4/0.25 Msucrose/0.2 mM CaCl2) at -700C until used.

Separation of inside-out vesicles was performed usingantibody-induced density perturbation as described (10, 19).Briefly, 1 mg of protein from CMVs was incubated with 0.5mg of anti-rat y-GT antibody in buffer A for 20 min at 370Cand then for 60 min at 0C. Excess antibody was removed bycentrifugation. Then, 0.5 mg of second antibody (anti-rabbitIgG antibody) was added and incubated for 20 min at 370C.Samples were layered on a continuous sucrose gradientconsisting of 5 ml of 20% (wt/vol) and 5 ml of 50% sucrosein 10 mM Hepes Tris, pH 7.4/0.2 mM CaC12 with 1 ml of60%sucrose as a cushion and centrifuged for 60 min at 17,000 xg. Fractions (1 ml) were collected and activities of y-GT andleucine aminopeptidase were measured. As reported (10, 19),

Abbreviations: BSP, sulfobromophthalein; y-GT, y-glutamyltrans-ferase; GSBSP, sulfobromophthaleinyl glutathione; GSDNP, dini-trophenyl glutathione; GSSG, oxidized glutathione; CMV, canalic-ular membrane vesicle; SMV, sinusoidal membrane vesicle; DIDS,4,4'-diisothiocyanostilbene-2,2'-disulfonate.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Temperature-dependent and ATP-dependent tauro-cholate transport by CMVs. Taurocholate transport in CMVs wasmeasured in the incubation medium containing 5 1LM [3H]tauro-cholate at 0C (squares) and at 370C (circles). Open symbols showtaurocholate transport into CMVs with 1.2 mM ATP and solidsymbols show transport without ATP. (Inset) Effect of mediumosmolarity on taurocholate transport. Transport activity was mea-sured in CMVs that had been treated for 30 min at 250C with 0-0.5M raffinose. Transport was performed in the incubation mediumadjusted to osmolality of the vesicle-containing buffer. Vesicle-associated taurocholate after a 20-s incubation was measured in thepresence (open circle) or absence (solid circle) of ATP. Values fortaurocholate uptake are expressed as pmol per mg per 20 s [mean +SEM (n = 3)]. In this and subsequent figures, unless indicated, theSEM value was less than the symbol size. osM, osmolar; prot.,protein.

two peaks of enzyme activity were detected in the samplethat had been treated with anti-y-GT. Vesicles were collectedfrom low- and high-density fractions and used for transportstudies. Vesicles recovered from the low-density fractionswere mainly oriented inside-out and vesicles from the high-density fractions were right side-out (10). A control experi-ment was performed as described above by replacing anti-yGT antibody with nonimmune rabbit serum. In the controlexperiment, only one peak of marker enzyme activity, whichwas in the low-density fraction, was detected.Marker enzymes were assayed as described (20-22). Pro-

tein concentration was determined by the method of Lowryet al. (23) using bovine serum albumin as a standard.

Transport Studies. The transport of taurocholate was mea-sured by rapid filtration (24). Unless otherwise mentioned,the incubation medium contained various concentrations oftaurocholate, 1.2 mM ATP, and an ATP regenerating system[3 mM creatine phosphate/creatine kinase (100 ug/ml)] in

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buffer B (10 mM Hepes-Tris, pH 7.4/0.25 M sucrose/10 mMMgCl2/0.2 mM CaCl2). Transport was initiated by adding 20Al of vesicles (40-80 ,ug of protein) suspended in buffer A to0.1 ml of the incubation medium that was preincubated for 5min at 370C. Samples of 20 A.l were removed as indicated anddiluted to 1 ml with ice-cold buffer B. Vesicles were filteredthrough glass microfiber filters (Whatman; 0.45-Am poresize) that were washed twice with 10 ml of ice-cold buffer B.Radioactivity on the filters was measured in a liquid scintil-lation spectrometer (Beckman; model LS 1801).

All experiments were repeated at least three times withdifferent preparations. Values are expressed as mean ± SEafter correction for radioactivity found on filters in theabsence of membrane vesicles.

RESULTSTaurocholate Transport by Membrane Vesicles. In the pres-

ence of 1.2 mM ATP, vesicle-associated taurocholate rapidlyincreased at 37°C to a maximum at 1 min followed by gradualdecrease to equilibrium at 30 min (Fig. 1). ATP-dependentvesicle-associated taurocholate was temperature-dependent;there was little increase in vesicle-associated taurocholate byATP at 0°C. The effect of increasing medium osmolality wasmeasured using medium containing various concentrations ofraffinose (Fig. 1 Inset). ATP-dependent vesicle-associatedtaurocholate decreased with increasing medium osmolality.In the absence of ATP, vesicle-associated taurocholate wasunaffected by increasing medium osmolality. These resultssuggest that taurocholate is transported into CMVs only inthe presence of ATP.The effect ofATP on taurocholate transport by SMVs was

also evaluated. SMVs exhibited sodium-dependent transportof taurocholate, as reported (2). SMVs did not reveal ATP-dependent taurocholate transport (Fig. 2).

Effect of Nucleotides on Taurocholate Transport by CMVs.ATP-dependent taurocholate transport by CMVs was mea-sured as a function of ATP concentration (Fig. 3A). Tauro-cholate transport was concentration-dependent and satura-ble. Uptake values followed Michaelis-Menten kinetics withan apparent Km of 0.67 + 0.11 mM (n = 3). Taurocholatetransport was determined after replacing ATP with variousnucleotides and nonhydrolyzable ATP analogs (Table 1).Among the nucleotide triphosphates tested, there was aspecific requirement for ATP. ADP marginally enhancedtransport. Nonhydrolyzable ATP analogs, such as adenosine5'-[,8y-methylene]triphosphate and adenosine 5'-[f3,y-imido]triphosphate, and other adenine nucleotides failed tostimulate taurocholate transport. These results indicate thathydrolysis of the y-phosphate of ATP is essential for trans-port.

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FIG. 2. Taurocholate transport by SMVs. SMVs were incubated in the incubation medium containing 5 ,uM [3H]taurocholate at 37°C.Additions to the incubation medium were as follows. (A) e, Incubation at 0°C, no additions; *, 0.1 M KCl; o, 0.1 M NaCl. (B) o, No addition;o, 1.2 mM ATP; A, 0.1 M KCI plus valinomycin at 50 ;&g/ml. Values are expressed as mean + SEM (n = 3).

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FIG. 3. Effects of ATP concentration and taurocholate concentration on ATP-dependent taurocholate transport by CMVs. (A) Incubationmedium contained 5 iL.M (3H]taurocholate, various concentrations of ATP, and an ATP-regenerating system. (B) CMVs (about 50 Ag of proteinper ml) were incubated with various concentrations of taurocholate in 0.12 ml of the incubation medium. After a 20-s incubation at 37TC,vesicle-associated radioactivity was measured. ATP-dependent taurocholate transport was obtained from the difference in the presence andabsence of ATP. Values for taurocholate transport are expressed as pmol per mg per 20 s (mean SEM; n = 3).

ATP-dependent taurocholate transport by CMVs exhibitedsaturation kinetics with respect to taurocholate concentra-tion (Fig. 3B). The apparent Km for taurocholate was 26 ± 1jxM (n = 3) and Vmax was 0.15 ± 0.01 nmol per mg of proteinper 20 s (n = 3).

Taurocholate Transport by Inside-Out CMVs. The CMVsused consist of ==90% right side-out vesicles and 10% inside-out vesicles (10, 19). To determine the sideness of vesiclesresponsible for ATP-dependent taurocholate transport, in-side-out vesicles were separated by antibody-induced densityperturbation. Since y-GT is exclusively localized on the outersurface ofthe canalicular membrane (19), anti-y-GT antibodywas used for the separation, as reported (10). As shown inFig. 4, inside-out vesicles, but not right side-out vesicles,showed ATP-dependent taurocholate transport.

Effect of Various Organic Compounds on ATP-DependentTaurocholate Transport. Because hydrolysis ofATP appearsto be essential for ATP-dependent taurocholate transport, theeffect of inhibitors of ATPase and organic anion carriers wasexamined. Since vanadate acts as a transitional state analogfor phosphate release from some ATPases, such as Na',K+-ATPase and Ca2+-ATPase (25), inhibition by vanadate sug-gests that ATPase activity is involved. As shown in Fig. 5,

Table 1. Effect of nucleotides and ATP analogs on taurocholatetransport in CMVs

Nucleotide-dependent transport,

Nucleotide pmol per mg per 20 s

ATPTTPUTPCTPGTPITPATPpp[CH2]pApp[NH]pAADPAMPAdenosine

11.5 ± 2.10.7 ± 0.1

-0.2 ± 0.31.2 ± 0.30.5 ± 0.40.8 ± 0.313.6 ± 1.60.6 ± 1.01.0 ± 0.44.1 ± 1.81.3 ± 0.70.9 ± 0.2

Relativetransport, %

1004.2 ± 0.5

-2.2 ± 0.51.7 ± 2.22.7 ± 1.94.1 ± 2.3

1004.7 ± 3.30.2 ± 0.2

21.6 ± 11.61.5 ± 1.01.2 ± 1.5

Transport was measured in incubation medium containing 5 A&M[3H]taurocholate and various agents at 1 mM without an ATP-regenerating system. After a 20-s incubation, vesicle-associatedradioactivity was measured. Nucleotide-dependent transport wasobtained from the difference in the presence and absence of thenucleotide. Values are expressed as mean ± SEM (n = 3).pp[CH2]pA, adenosine 5'-[,',y-methylene]triphosphate; pp[NH]pA,adenosine 5'-[,3,y-imido]triphosphate.

ATP-dependent taurocholate transport was inhibited by van-adate in a dose-dependent manner. Half-maximal inhibitionwas obtained at about 20 ,uM. These results suggest arequirement for ATPase activity and/or phosphorylated in-termediates by the putative carrier.To characterize further the mechanism of ATP-dependent

taurocholate transport, we studied the effect of variousinhibitors. As shown in Table 2, transport was partiallyinhibited by oligomycin and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), an anion transport inhibitor (7), and wascompletely inhibited by sulfinpyrazone and probenecid, in-hibitors of other organic anion transport systems (26). Stro-phanthidin, ouabain, and pyrocarbonate, which partiallyinhibits ecto-ATPase (S. H. Lin, personal communication),had little effect.

Inhibition of taurocholate transport by other organic com-pounds was also examined (Table 3). Cholate and glyco-cholate inhibited transport. Although daunomycin (10) andvarious glutathione S-conjugates (11-13) are transported intoCMVs by ATP-dependent mechanisms, daunomycin, GSSG,bilirubin diglucuronide, and GSDNP had no inhibitory effecton taurocholate transport. BSP and GSBSP inhibited tauro-cholate transport. These results suggest that taurocholate istransported into CMVs by a carrier that is different from

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FIG. 4. Taurocholate transport into inside-out vesicles and rightside-out CMVs. Taurocholate transport was measured with vesiclescollected from low-density fractions, which were mainly oriented inside-out (circles), and with vesicles from high-density fractions, which wereright side-out vesicles (squares). Vesicle-associated taurocholate wasdetermined in the presence (open symbols) or absence (solid symbols) ofATP and an ATP-regenerating system. The concentration of tauro-cholate was 10 1tM. Other conditions were as described in Fig. 1. Valuesare expressed as mean ± SEM (n = 4).

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port by CMVs. Vesicles were preincubated with the indicatedconcentration of sodium vanadate for 10 min at 37°C. Transport wasstarted by adding preincubated vesicles to the incubation mediumthat contained the indicated concentration of sodium vanadate and 5MM [3H]taurocholate. After a 20-s incubation at 37°C, vesicle-associated radioactivity was measured. ATP-dependent tauro-cholate transport was obtained from the difference in the presenceand absence of ATP and an ATP-regenerating system. Values wereexpressed in percentage of the ATP-dependent taurocholate trans-port activity without vanadate (11 ± 1 pmol per mg of protein per 20s). Values are expressed as mean ± SEM (n = 3).

P-glycoprotein (10) and from the putative nonbile acid or-ganic anion carrier (11-13).

Filipin was used to characterize further taurocholate trans-port and differences among transport mechanisms for dauno-mycin, BSP, and taurocholate. Filipin is an antifungal anti-biotic that forms a complex with cholesterol; incubation ofmembrane vesicles with filipin decreases membrane choles-terol content. Incubation with filipin inhibited Na+-dependent taurocholate uptake by endoplasmic reticulumand Golgi apparatus (27). As shown in Fig. 6, ATP-dependenttaurocholate transport by CMVs was inhibited by filipin,although ATP-dependent daunomycin and BSP transportwere not inhibited. Half-maximal inhibition of taurocholatetransport was obtained at 0.023 mg/mg of protein. This resultindicates that membrane cholesterol is required for ATP-dependent taurocholate transport and provides further evi-dence that the taurocholate transport system differs from the

Table 2. Effect of various inhibitors on ATP-dependenttaurocholate transport in CMVs

Taurocholate Relativetransport, transport,

Inhibitor pmol per mg per 20 s %Control 8.6 ± 0.3 100DIDS (0.2 mM) 4.2 ± 0.2 56 ± 6Pyrocarbonate (0.1 mM) 9.9 ± 2.8 108 ± 9Sulfinpyrazone (1 mM) 0.07 ± 0.06 3 ± 2Control 10.0 ± 0.5 100Oligomycin (20 ,g/ml) 2.4 ± 0.4 23 ± 4Sodium azide (1 mM) 7.4 ± 0.7 76 ± 11N-Ethylmaleimide (20 AM) 10.1 ± 1.9 105 ± 24Amiloride (0.2 mM) 9.4 ± 2.7 91 ± 24Probenecid (5 mM) -3.4 ± 0.05 -3 ± 4Ouabain (1 mM) 9.4 ± 0.5 91 ± 5Strophanthidin (1 mM) 10.6 ± 2.0 102 ± 11

Incubation medium contained, in a final volume of 0.12 ml, 5 AM[3H]taurocholate, various inhibitors, vesicles (40-60 Mg of protein),and 1.2 mM ATP with an ATP-regenerating system. After a 20-sincubation, vesicle-associated radioactivity was measured. ATP-dependent taurocholate transport was calculated by subtractingvalues obtained in the absence of ATP. The final concentration ofvarious inhibitors in the incubation medium is shown in the table.Values are expressed as mean ± SEM (n = 3).

Table 3. Inhibition of ATP-dependent taurocholate transport byorganic compounds

Transport, RelativeCompound pmol per mg per 20 s transport, %

Control 10.4 ± 0.1 100Glycocholate (10 MM) 2.2 ± 0.7 22 ± 7Cholate (10 MM) 3.1 ± 0.5 31 ± 5BSP (25MM) 1.9 ± 0.5 18 ± 3GSBSP (25 MM) 6.1 ± 0.3 58 ± 2GSDNP (25 MM) 10.6 ± 1.4 102 ± 14GSSG (25 ,uM) 8.3 ± 1.1 82 ± 10GSSG (3 mM) 13.5 ± 1.3 130 ± 13Bilirubin diglucuronide(10MAM) 7.6 ± 0.8 74 ± 8

Daunomycin (25 MM) 8.7 ± 0.6 86 ± 11Incubation medium contained 5 AuM [3H]taurocholate, 1.2 mM

ATP with an ATP-regenerating system, and the indicated concen-tration of various organic compounds. After a 20-s incubation,vesicle-associated radioactivity was measured. ATP-dependenttransport was obtained from the difference in the presence andabsence of ATP. Values are expressed as mean ± SEM (n = 3).

ATP-dependent systems for daunomycin and nonbile acidorganic anions.

DISCUSSIONTaurocholate transport across the canalicular membrane is acarrier-mediated process (8). Transport is driven by theoutside-positive membrane potential (8, 9). In this report,another mechanism of taurocholate transport that involvesATP-dependent transport is demonstrated.ATP dependence was confirmed by the following obser-

vations. (i) ATP-dependent taurocholate transport revealedan overshoot phenomenon (Figs. 1 and 4), which is exhibitedby several ATP-dependent processes (10-13). The mecha-nism is not known but nonionic diffusion oftaurocholate fromthe vesicles is believed to be partially responsible (10). (ii)Temperature and, in particular, osmolality dependence ofATP-dependent taurocholate transport indicate transmem-brane transport (Fig. 1). (iii) The saturable process followsMichaelis-Menten kinetics with respect to ATP and tauro-

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FIG. 6. Effect offilipin on ATP-dependent taurocholate transportby CMVs. Vesicles were preincubated with the indicated concen-tration of filipin for 10 min at 37°C. Transport was started by addingthe preincubated vesicles to incubation medium containing 5 ,uM[3H]taurocholate. After a 20-s incubation at 37°C, vesicle-associatedradioactivity was measured, ATP-dependent taurocholate transport(o) was obtained from the difference in the presence and absence ofATP and an ATP-regenerating system. For ATP-dependent dauno-mycin transport (o) and BSP transport (A), [3H]taurocholate wasreplaced by 10 MM [3H]daunomycin or by 0.2 MM "S-labeled BSP.Values were expressed in percentage of the ATP-dependent trans-port activity without filipin. The values for 100%6 activities oftaurocholate, daunomycin, and BSP transport were 11 ± 1, 240 ± 10,and 26 ± 2 pmol per mg of protein per 20 s, respectively. Values areexpressed as mean + SEM (n = 3).

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cholate, indicating a carrier-mediated system (Fig. 3). (iv)ATP was specifically required in promoting taurocholatetransport among various nucleotides and nonhydrolyzableATP analogs (Table 1). (v) ATP-dependent taurocholatetransport was inhibited by vanadate in a dose-dependentmanner (Fig. 5).

Inhibition of ATP-dependent taurocholate transport bycholate and glycocholate and by anion-transport inhibitors,such as DIDS, probenecid, and sulfinpyrazone, suggestssubstrate specificity of the system (Tables 2 and 3). Twoother ATP-dependent transport systems have been describedin the canalicular membrane: P-glycoprotein (10) and a trans-port system for glutathione S-conjugates and other nonbileacid organic anions (11-13). Daunomycin (which is trans-ported by P-glycoprotein), GSSG, bilirubin diglucuronide,and GSDNP did not inhibit ATP-dependent taurocholatetransport. Although BSP and GSBSP inhibited taurocholatetransport, the following evidence suggests that the ATP-dependent taurocholate transport system differs from theBSP transport system and that inhibition by BSP and GSBSPprobably represents binding as reported in basolateral mem-branes (7). (i) Taurocholate and cholate inhibit ATP-dependent taurocholate transport (Table 3) but not ATP-dependent BSP transport (data not shown). (ii) CMVs fromTR- mutant rats, which lack ATP-dependent transport ofBSP and GSBSP (11), retain ATP-dependent taurocholatetransport (unpublished data). (iii) Filipin inhibited ATP-dependent taurocholate transport but not ATP-dependentBSP transport (Fig. 6). Thus, the ATP-dependent tauro-cholate transport system in CMVs is distinct from the ATP-dependent system for nonbile acid organic anions.A bile acid transport system in the canalicular membrane

was initially demonstrated by kinetic studies using CMV (8).This system exhibited transstimulation and was inhibited byconjugated dihydroxy bile acids, DIDS, N-ethylmaleimide,and high concentrations of GSSG, suggesting the possibleinvolvement of protein SH groups (7-9). Transport wasdriven by the intracellular negative membrane potential (ap-proximately -30 to -40 mV) (7-9, 28). Based on studies withpolyclonal antibodies and reconstitution into proteolipo-somes, a 100-kDa glycoprotein has been proposed to be thecarrier (29-31).Whether or not the ATP-dependent and membrane poten-

tial-dependent systems share the same carrier is not known.Both systems are inhibited by cholate, glycocholate, andDIDS. However, the ATP-dependent system is not sensitiveto N-ethylmaleimide and is not inhibited by high concentra-tions ofGSSG (Tables 2 and 3). These results suggest that theATP and membrane potential-dependent transport systemsmay be different.The physiological role and interrelationship between two

taurocholate transport systems are not known. Although anexperimentally produced increase in membrane potentialincreased canalicular bile secretion in isolated hepatocytecouplets (32), the role of the ATP-dependent system was notevaluated. The apparent Km for ATP-dependent system (26gM) is lower than that for the potential-dependent system (43,uM, ref. 8), whereas Vma,, for each system is comparable[0.15 nmol per mg of protein per 20 s and 0.22 nmol per mgof protein per 20 s (8), respectively]. Therefore, both systemsmay function physiologically. Because bile acid secretion isthe major determinant ofbile flow, defects in this system may

be anticipated to result in impaired bile secretion (cholesta-sis).

This work was supported in part by Grant DK 35652 from theNational Institutes of Health (to I.M.A.).

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