Reconstitution of Bile Acid Transport in a Heterologous Cell by Cotransfection of Transporters for Bile Acid Uptake and Efflux
1997; Elsevier BV; Volume: 272; Issue: 29 Linguagem: Inglês
10.1074/jbc.272.29.18290
ISSN1083-351X
AutoresClaudia Sippel, Paul A. Dawson, Tianxiang Shen, David H. Perlmutter,
Tópico(s)Drug Solubulity and Delivery Systems
ResumoThe rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 (CBATP) is a 110-kDa transmembrane phosphoglycoprotein that is thought to have bile acid efflux, ecto-ATPase, and cell adhesion properties. Its extracellular amino-terminal domain is highly homologous to carcinoembryonic antigen (CEA), a glycophosphatidyl inositol-anchored membrane protein with cell adhesion properties and a marker for adenocarcinoma. In the current study, we examined the possibility of more clearly defining the role of CBATP in bile acid efflux by cotransfecting a heterologous cell, the COS cell, with cDNAs for a bile acid importer, the ileal bile acid transporter (IBAT), as well as for CBATP. The results show that when IBAT mediates uptake of [3H]taurocholate to a level 20-fold higher than that achieved previously by nonspecific pinocytosis, CBATP mediates time-, temperature- and concentration-dependent efflux. Efflux of [3H]taurocholate mediated by CBATP in the cotransfected COS cells is saturable and has curvilinear kinetic characteristics (V max = 400 pmol/mg protein/min,K m = 70 μm). It is inhibited by 4,4′-diisothiocyanostilbene-2,2-disulfonic acid and dependent on ATP but not dependent on membrane potential. Although CEA could not mediate bile acid efflux in COS cells cotransfected with IBAT and CEA, efflux of [3H]taurocholate was detected in COS cells cotransfected with IBAT and a chimeric molecule having the carboxyl-terminal tail and membrane spanning domain of CBATP and the amino-terminal extracellular tail of CEA. Taken together, these data provide further evidence that CBATP confers bile acid efflux properties on heterologous cells and that its cytoplasmic tail and membrane spanning segment are integral to this property. The data also establish a model system for more clearly defining the molecular determinants of bile acid transport mediated by this molecule. The rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 (CBATP) is a 110-kDa transmembrane phosphoglycoprotein that is thought to have bile acid efflux, ecto-ATPase, and cell adhesion properties. Its extracellular amino-terminal domain is highly homologous to carcinoembryonic antigen (CEA), a glycophosphatidyl inositol-anchored membrane protein with cell adhesion properties and a marker for adenocarcinoma. In the current study, we examined the possibility of more clearly defining the role of CBATP in bile acid efflux by cotransfecting a heterologous cell, the COS cell, with cDNAs for a bile acid importer, the ileal bile acid transporter (IBAT), as well as for CBATP. The results show that when IBAT mediates uptake of [3H]taurocholate to a level 20-fold higher than that achieved previously by nonspecific pinocytosis, CBATP mediates time-, temperature- and concentration-dependent efflux. Efflux of [3H]taurocholate mediated by CBATP in the cotransfected COS cells is saturable and has curvilinear kinetic characteristics (V max = 400 pmol/mg protein/min,K m = 70 μm). It is inhibited by 4,4′-diisothiocyanostilbene-2,2-disulfonic acid and dependent on ATP but not dependent on membrane potential. Although CEA could not mediate bile acid efflux in COS cells cotransfected with IBAT and CEA, efflux of [3H]taurocholate was detected in COS cells cotransfected with IBAT and a chimeric molecule having the carboxyl-terminal tail and membrane spanning domain of CBATP and the amino-terminal extracellular tail of CEA. Taken together, these data provide further evidence that CBATP confers bile acid efflux properties on heterologous cells and that its cytoplasmic tail and membrane spanning segment are integral to this property. The data also establish a model system for more clearly defining the molecular determinants of bile acid transport mediated by this molecule. The net vectorial transport of bile acids into the biliary drainage system is the major determinant of bile secretion. Transport across the canalicular domain of the hepatocyte represents the rate-limiting step in this system. Studies in canalicular membrane vesicles have indicated that canalicular bile acid efflux is predominantly driven by ATP, but electrochemical membrane potential may also drive canalicular bile acid transport to a certain extent. Most data suggest that several distinct transporters, or transport systems, are involved (1Blitzer B.L. Boyer J.L. Gastroenterology. 1982; 82: 346-357Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 2Scharschmidt B.F. van Dyke R.W. Gastroenterology. 1983; 85: 1199-1214Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 3Hardison W.G.M. Wood C.A. Am. J. Physiol. 1978; 235: E158-E164PubMed Google Scholar, 4Dumont M. Erlinger S. Uchamn S. Gastroenterology. 1980; 79: 82-89Abstract Full Text PDF PubMed Scopus (194) Google Scholar, 5Forker E.L. Annu. Rev. Physiol. 1977; 39: 323-347Crossref PubMed Scopus (122) Google Scholar, 6Gaitman Z.C. Arias I.M. Physiol. Rev. 1995; 75: 261-275Crossref PubMed Scopus (65) Google Scholar). The rat liver canalicular bile acid transport protein/ecto-ATPase/cell CAM 105 (CBATP) 1The abbreviations used are: CBATP, canalicular bile acid transport protein; CEA, carcinoembryonic antigen; DMEM, Dulbecco's modified Eagle's medium; IBAT, ileal bile acid transport protein; DIDS, 4,4′-diisothiocyanostilbene-2,2-disulfonic acid; ATPγS, adenosine 5′-O-(thiotriphosphate). is one candidate for bile acid efflux activity at the canalicular membrane of hepatocytes. It is a ∼110-kDa phosphoglycoprotein localized to the canalicular domain of hepatocytes. It was purified by bile acid affinity chromatography from detergent-solubilized rat liver canalicular membrane vesicles (7Sippel C.J. Ananthanarayanan M. Suchy F.J. Am. J. Physiol. 1990; 21: G728-G737Google Scholar). Internal amino acid sequence analysis revealed it to be identical to the rat liver ecto-ATPase and to the cell adhesion molecule cell CAM 105. It has a carboxyl-terminal cytoplasmic tail of 71 amino acids, a single membrane-spanning domain, and a large extracellular amino-terminal tail of 423 amino acids, which has extensive homology with carcinoembryonic antigen (CEA) and the immunoglobulin supergene family. Transfection studies have shown that CBATP mediates ecto-ATPase, cell adhesion, and bile acid efflux activities in heterologous COS cells (8Sippel C.J. Suchy F.J. Ananthanarayanan M. Perlmutter D.H. J. Biol. Chem. 1993; 268: 2083-2091Abstract Full Text PDF PubMed Google Scholar, 9Cheung P.H. Thompson N.L. Earley K. Culic O. Hixson D. Lin S.-H. J. Biol. Chem. 1993; 268: 6139-6146Abstract Full Text PDF PubMed Google Scholar). Mutagenesis studies have shown that R98 within an ATPase consensus sequence at the extreme amino terminus is required for ecto-ATPase activity and that the 108 amino acid amino-terminal domain is required for cell adhesion activity (10Sippel C.J. McCollum M.J. Perlmutter D.H. J. Biol. Chem. 1994; 269: 2820-2826Abstract Full Text PDF PubMed Google Scholar, 11Cheung P.H. Luo W. Qiu Y. Zhang X. Earley K. Millirans P. Lin S.-H. J. Biol. Chem. 1993; 268: 24303-24310Abstract Full Text PDF PubMed Google Scholar). Deletion of the cytoplasmic tail is associated with loss of bile acid efflux, ecto-ATPase, and cell adhesion activities, even though the protein is appropriately localized to the external surface of the plasma membrane and can bind ATP (8Sippel C.J. Suchy F.J. Ananthanarayanan M. Perlmutter D.H. J. Biol. Chem. 1993; 268: 2083-2091Abstract Full Text PDF PubMed Google Scholar, 9Cheung P.H. Thompson N.L. Earley K. Culic O. Hixson D. Lin S.-H. J. Biol. Chem. 1993; 268: 6139-6146Abstract Full Text PDF PubMed Google Scholar). Site-directed mutagenesis of phosphorylation consensus sequences in the cytoplasmic tail shows that protein kinase C-dependent phosphorylation of Ser503 is required for bile acid efflux activity, that tyrosine kinase-dependent phosphorylation of Tyr488 regulates bile acid efflux activity, but neither phosphorylation is necessary for ecto-ATPase activity. Finally, bile acid efflux activity of CBATP was found to be dependent on ATP, particularly extracellular ATP, but not on its own ecto-ATPase activity (12Sippel C.J. Fallon R.J. Perlmutter D.H. J. Biol. Chem. 1994; 269: 19539-19545Abstract Full Text PDF PubMed Google Scholar). In these previous studies, the bile acid efflux activity of CBATP was demonstrated in transfected COS cells by first loading the cells with [3H]taurocholate via nonspecific pinocytosis using the approach that had been pioneered in studies of efflux of chemotherapeutic agents by the multidrug resistance gene products (MDR) (8Sippel C.J. Suchy F.J. Ananthanarayanan M. Perlmutter D.H. J. Biol. Chem. 1993; 268: 2083-2091Abstract Full Text PDF PubMed Google Scholar). To get sufficient nonspecific uptake, the membrane potential of the transfected COS cells had to be clamped pharmacologically with valinomycin (8Sippel C.J. Suchy F.J. Ananthanarayanan M. Perlmutter D.H. J. Biol. Chem. 1993; 268: 2083-2091Abstract Full Text PDF PubMed Google Scholar). Although this system allowed us to show that CBATP had bile acid efflux activity and that this could be proven by a number of genetic criteria, the assay was cumbersome and was not amenable to studying the role of membrane potential in bile acid efflux. To develop a more physiological system and a system that could be more easily manipulated pharmacologically, we have established a model system in which uptake of [3H]taurocholate is mediated by transfection of a cloned bile acid importer, IBAT, and efflux is assayed by cotransfection of CBATP cDNA. Wild-type and mutant rat CBATP constructs have been described previously (8Sippel C.J. Suchy F.J. Ananthanarayanan M. Perlmutter D.H. J. Biol. Chem. 1993; 268: 2083-2091Abstract Full Text PDF PubMed Google Scholar, 10Sippel C.J. McCollum M.J. Perlmutter D.H. J. Biol. Chem. 1994; 269: 2820-2826Abstract Full Text PDF PubMed Google Scholar, 11Cheung P.H. Luo W. Qiu Y. Zhang X. Earley K. Millirans P. Lin S.-H. J. Biol. Chem. 1993; 268: 24303-24310Abstract Full Text PDF PubMed Google Scholar). This includes pExp3, R98ACBATP, Y488F-CBATP, T502,S503A-CBATP, and truncated CBATP. The hamster IBAT construct (IBAT-44 final) has also been described previously (13Wong M.H. Oelkers P. Craddock A.L. Dawson P.A. J. Biol. Chem. 1994; 269: 1340-1347Abstract Full Text PDF PubMed Google Scholar). The CEA cDNA (gcg:humcea; Ref. 14Barnett T. Goebel S.J. NothDurft M.A. Elting J.J. Genomics. 1988; 3: 59-66Crossref PubMed Scopus (41) Google Scholar), kindly provided by Thomas Barnett, was subcloned into theHindIII-XbaI site of pCDM8. Three new constructs, R98A CEA, CBATP-CEA chimera, and CBATP-R98ACEA chimera, were generated using the polymerase chain reaction overlap extension technique (15Higuchi R. Innis M.A. Gelfand D.H. Snisky J.W. White T.H. PCR Protocols. Academic Press, Inc., San Diego1990: 177-180Google Scholar) and then subcloned into the pCDM8 vector. For R98ACEA, the outside primers corresponded with nucleotides 85–100 and 993–1012 of CEA together with a spacer of four nucleotides, a newEcoRI restriction site, and a new BamHI restriction site (5′-TCATGAATTCAGAGGAGGACAGAGC-3′; 5′-GGCACGTATAGGATCCACTA-3′). The inside primers corresponded to nucleotides 393–416 of CEA (5′-CGCATACAGTGGTGCAGAGATAAT-3′; 5′-ATTATCTCTGCACCACTGTATGCG-3′). The resulting polymerase chain reaction fragment was subcloned into wild-type CEA in the pCDM8 vector, which had been digested previously withEcoRI/BamHI and purified away from the wild-type internal fragment. For the CEA-CBAT chimera, the outside primers corresponded to nucleotides 1563–1585 of CEA and nucleotides 1628–1651 of CBATP together with new EcoRI and XbaI restriction sites, and the inside primers corresponded to nucleotides 2122–2136 of CEA (five amino acids just external to the membrane insertion site of CEA) and nucleotides 1316–1330 of CBATP (transmembrane domain of CBATP): outside primers (5′-CAGTGGCCACAGCAGGACTACAG-3; 5′-GAATTCTCTAGACTGGTGCAGTCAGCAGGACAGACA-3′); inside primers (5′-TGAGAGGCCAGAATTGACTGTGATGCTCTT-3′; 5′-AAGAGCATCACAGTCAATTCTGGCCTCTCA-3′). The resulting 0.95-kilobase polymerase chain reaction fragment was digested withBalI and XbaI and cloned into pBluescript together with a 1.5-kilobase BalI/EcoRI partial digest of wild-type CEA. The resulting insert was removed atHindIII/XbaI to be subcloned into pCDM8. A similar strategy was used for the CBATP-R98ACEA chimera using the R98ACEA construct described above. In each case, the constructs were characterized by restriction map analysis (16Hanahan D. J. Mol. Biol. 1988; 166: 557-580Crossref Scopus (8216) Google Scholar, 17Birnboin H.C. Boly J. Nucleic Acids Res. 1979; 7: 1513-1523Crossref PubMed Scopus (9908) Google Scholar) and by dideoxynucleotide sequencing (18Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4767-4771Crossref PubMed Scopus (1687) Google Scholar) to confirm the construct and to exclude polymerase chain reaction sequence artifacts. COS 1 cells were transfected by the DEAE-dextran method and used for experimental purposes 48 h after transfection (19Cullen B.R. Berger S.L. Kimmel A.R. Methods in Enzymology. 152. Academic Press, Inc., San Diego1987: 692-693Google Scholar). In specific experiments, cellular ATP was depleted by incubating transfected COS cells for 20 min at 37 °C in DMEM without glucose but supplemented with 20 mm2-deoxyglucose and 10 mm sodium azide. Under these conditions, cellular concentrations of ATP could be lowered from 800–1200 μm to 6–8 μm, as determined by the ATP luciferase assay (20Yih L.H. Hunag H. Jan K.Y. Lee T. Cell Biol. Int. Rep. 1991; 15: 253-264Crossref PubMed Scopus (41) Google Scholar). Results were normalized for protein concentration as determined by the Lowry assay (21Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Under these conditions, cell viability as determined by trypan blue exclusion (22Blitzer B. Ratoosh S.L. Donovan C.B. Boyer J.L. Am. J. Physiol. 1982; 6: G48-G53Google Scholar) was not significantly different between these depleted cells and undepleted cells (data not shown). Transfected Cos cells were incubated with DMEM supplemented with [3H]taurocholate in excess. At the end of the uptake period, cell monolayers were lysed in 1n NaOH, and the cell lysates were subjected to scintillation counting. In separate experiments, the time, temperature, and concentration of [3H]taurocholate were varied to determine the optimal conditions for uptake. Transfected Cos cells were incubated with [3H]taurocholate under optimal conditions for uptake. At the end of the uptake period, monolayers were washed extensively in PBS and incubated for specified time intervals in DMEM alone for the efflux period. In specified experiments, 5 μm ATP, 1 mm DIDS, and/or unlabeled taurocholate were added. Extracellular medium was harvested, and cell monolayers were lysed in 1 n NaOH. The extracellular medium and cell lysates were then subjected to scintillation counting. Counts in the extracellular medium were converted to picomoles on the basis of the specific activity of the initial [3H]taurocholate and then plotted as pmoles/milligram protein/minute. Kinetic data (K m and V max) were determined on the basis of the resulting curves. Ecto-ATPase activity was measured by a method described previously (23Lin S.-H. Guidotti G. J. Biol. Chem. 1989; 264: 14408-14414Abstract Full Text PDF PubMed Google Scholar) 48 h after transfection. For Western blot analysis, antibody to CBATP was used in a protocol described previously (7Sippel C.J. Ananthanarayanan M. Suchy F.J. Am. J. Physiol. 1990; 21: G728-G737Google Scholar). Methods described previously were also used for studies of biosynthesis (24Perlmutter D.H. Punsal P.I. J. Biol. Chem. 1988; 263: 16499-16503Abstract Full Text PDF PubMed Google Scholar), cell surface iodination (25Lederkremer G.Z. Lodish H.F. J. Biol. Chem. 1991; 266: 1237-1244Abstract Full Text PDF PubMed Google Scholar), and immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis/fluorography (24Perlmutter D.H. Punsal P.I. J. Biol. Chem. 1988; 263: 16499-16503Abstract Full Text PDF PubMed Google Scholar). We determined the conditions under which uptake of [3H]taurocholate was saturated in COS cells transfected with IBAT alone or IBAT and CBATP cDNA together. To determine the duration of time necessary for saturation of uptake, transfected COS cells were incubated at 22 °C with [3H]taurocholate, 400 μm, for several different time intervals (Fig.1 A). Cells were lysed in 1 n NaOH and subjected to scintillation counting. Uptake was time-dependent, reaching a plateau within 20 min. There was no significant difference between cells transfected with IBAT alone and cells transfected with IBAT and CBATP together. Then the transfected COS cells were incubated for 20 min with [3H]taurocholate, 400 μm, at several different temperatures (Fig. 1 B). Uptake was temperature-dependent, reaching a plateau at 22 °C. There was no significant difference between cells transfected with IBAT alone and those transfected with IBAT and CBATP together. Next, the transfected COS cells were incubated for 20 min at 22 °C in [3H]taurocholate in several different concentrations (Fig. 1 C). The results show that uptake was concentration-dependent, reaching a plateau between 200 and 400 μm. In COS cells transfected with IBAT and CBATP, there was a minimal decrease in the amount of uptake as compared with COS cells transfected with IBAT alone. This is probably due to efflux mediated by cotransfected CBATP. Efflux mediated by cotransfected CBATP does not have a more significant effect in Fig. 1 C or any effect in Fig. 1, A and B, because these experiments were done in the absence of exogenous ATP. Because efflux mediated by CBATP is stimulated by exogenous ATP (see Fig. 3), this means that there will be minimal efflux mediated by CBATP during uptake studies done in the absence of exogenous ATP. Even when exogenous ATP is present, CBATP pumps [3H]taurocholate out of cells less efficiently than IBAT pumps [3H]taurocholate into cells. For uptake mediated by IBAT, K m ≅ 23 μm and V max ≅ 396 pmol/mg protein/min (13Wong M.H. Oelkers P. Craddock A.L. Dawson P.A. J. Biol. Chem. 1994; 269: 1340-1347Abstract Full Text PDF PubMed Google Scholar), and for efflux mediated by CBATP in the presence of exogenous ATP, K m ≅ 70 μm andV max ≅ 400 pmol/mg protein/min (Fig.2 A). When uptake of [3H]taurocholate was assayed in the presence of exogenous ATP (5 mm), there was ∼40–50% reduction in uptake after 60 min in COS cells cotransfected with IBAT and CBATP as compared with COS cells transfected with IBAT alone (Fig. 1 D).Figure 3Effect of ATP on bile acid efflux in cotransfected COS cells. Cells were cotransfected with IBAT and CBATP cDNA and studied 48 h later. At that time, the cells were incubated for 20 min at 22 °C with 400 μm[3H]taurocholate to load the cells as well as 20 mm 2-deoxyglucose and 10 mm sodium azide to deplete cellular ATP. Previous studies have shown that these conditions severely deplete cellular ATP levels but do not affect cell viability or one function of CBAT, its ecto-ATPase activity (20Yih L.H. Hunag H. Jan K.Y. Lee T. Cell Biol. Int. Rep. 1991; 15: 253-264Crossref PubMed Scopus (41) Google Scholar). These conditions did not significantly alter uptake of [3H]taurocholate (data not shown). The cells were then washed and incubated for 5 min at 22 °C with fresh unlabeled medium in the absence or presence of 1 mm DIDS and in absence or presence of ATP in several different concentrations. Results are reported as mean ± 1.0 S.D. (bars).K m ≅ 10 μm;V max = 300 pmol/mg protein/min.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Efflux of [3H]taurocholate in transfected COS cells. Cells were transfected with IBAT cDNA alone, cotransfected with both IBAT and CBATP cDNA, or cotransfected with IBAT and the T502A, S503A mutant CBATP.A, concentration dependence. After 48 h, the cells were incubated for 60 min at 22 °C in DMEM supplemented with [3H]taurocholate in several different concentrations. The cells were then washed and incubated for 5 min at 22 °C in medium supplemented with 5 mm ATP in the absence or presence of 1 mm DIDS. The medium was then harvested; the cells were homogenized, and each was subjected to scintillation counting. There was no significant difference in the amount of counts present in the cells at the end of the uptake for cells transfected with IBAT alone (▪), IBAT and CBATP (•), or IBAT and T502A, S503A mutant CBATP (▴). V max = 400 pmol/mg protein/min,K m = 70 μm. B, time dependence. After 48 h, the cells were incubated for 60 min at 22 °C with DMEM supplemented with 400 μm[3H]taurocholate. The cells were then washed and incubated at 22 °C in DMEM supplemented with unlabeled taurocholate (400 μm), 5 mm ATP in the absence (•) or presence (▴) of 1 mm DIDS for several different time intervals up to 10 min. The extracellular medium was then harvested; the cells were homogenized, and each was subjected to scintillation counting. The results are reported as relative percentage by comparing the counts in the specified sample to the total amount of counts taken up by the cells after 60 min of uptake. The results of three samples at each time point are reported as mean ± 1.0 S.D. (bars).IC, cell monolayers; EC, extracellular medium.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Taken together, the experiments in Fig. 1 (A–C) show that uptake reaches a plateau at 200–400 μm in the cotransfected cells and establish a time of 20 min, temperature of 22 °C, and concentration of [3H]taurocholate of 400 μm as optimal for the subsequent studies. In each case, the uptake of [3H]taurocholate was 20-fold or more greater in COS cells transfected with IBAT alone or both IBAT and CBATP than untransfected COS cells and COS cells transfected with CBATP alone and pharmacologically clamped with valinomycin (8Sippel C.J. Suchy F.J. Ananthanarayanan M. Perlmutter D.H. J. Biol. Chem. 1993; 268: 2083-2091Abstract Full Text PDF PubMed Google Scholar). Next, we examined the possibility that CBATP mediated bile acid efflux under these conditions. For Fig. 2 A, COS cells were transfected with IBAT alone, cotransfected with IBAT and CBATP, or cotransfected with IBAT and a mutant CBATP (T502A, S503A-CBATP). Our previous studies had shown that this mutant CBATP did not undergo protein kinase C-mediated phosphorylation and lacked bile acid efflux activity, even though it was appropriately targeted to the external surface of the plasma membrane (12Sippel C.J. Fallon R.J. Perlmutter D.H. J. Biol. Chem. 1994; 269: 19539-19545Abstract Full Text PDF PubMed Google Scholar). After 48 h, the cells were incubated at 22 °C for 60 min with [3H]taurocholate in several different concentrations. At the end of this time interval, the cells were washed extensively and incubated at 22 °C for 5 min in the absence of [3H]taurocholate, in the presence of ATP, and in the absence or presence of DIDS. Radioactivity appearing in the extracellular medium was determined by scintillation counting. The results show that there was DIDS-sensitive, concentration-dependent, and saturable efflux only in cells cotransfected with IBATP and CBATP, not in cells transfected with IBAT alone or cotransfected with IBAT and mutant T502A, S503A-CBATP. Efflux had curvilinear characteristics, with a V max of 400 pmol/mg protein/min and aK m of 70 μm. These values are similar to those obtained using our previous assay system. These values are also similar to values that have been reported previously in canalicular vesicle studies (17Birnboin H.C. Boly J. Nucleic Acids Res. 1979; 7: 1513-1523Crossref PubMed Scopus (9908) Google Scholar). For the studies shown in Fig.2 A, we also measured radioactivity remaining in cell lysates. The results were plotted as pmoles/mg protein/minuteversus concentration of taurocholate and showed that disappearance from the cell had almost identical curvilinear characteristics, K m and V maxvalues (data not shown) as observed in Fig. 2 A for appearance in the extracellular medium. In both cases, however, we did not determine what proportion of taurocholate that is in the cells is freely available for efflux. Thus, the K m andV max values reported here must be considered estimates which apply only if all the taurocholate in the cell is freely available for efflux. Next, we examined the time course of efflux mediated by CBATP (Fig.2 B). COS cells transfected with IBAT alone or cotransfected with IBAT and CBATP were incubated for 60 min at 22 °C with [3H]taurocholate 400 μm. At the end of this time interval, the cells were washed extensively and then incubated at 22 °C for several different time intervals in medium supplemented with unlabeled taurocholate (400 μm) and 5 mmATP in the absence or presence of 1 mm DIDS. At the end of each time interval, the extracellular medium was harvested, and the cell monolayers were lysed for analysis by scintillation counting. Results are reported as a relative percentage using counts present in the cell lysate at time 0 as arbitrarily designated 100%. There was no difference in the counts present in the cell lysates at time 0 for cells transfected with IBAT alone or cells cotransfected with IBAT and CBATP. For cells cotransfected with IBAT and CBATP, the results show that there is time-dependent disappearance of radioactivity from the cells between time 0 and 5 min, coincident with the appearance of radioactivity in the extracellular medium. Almost 80% of the initial radioactivity has disappeared from the cells by 5 min of the chase period, and a similar percentage has appeared in the extracellular medium. The majority of this disappearance from cell monolayers and appearance in extracellular medium is DIDS-sensitive. There is, however, some radioactivity that leaks from the cells in the presence of DIDS. Interestingly, this DIDS-insensitive fraction has different kinetics, reaching a plateau within 3 min. For cells transfected with IBAT alone, there is some time-dependent disappearance of radioactivity from the cells and appearance of radioactivity in the extracellular medium. It is much less than that observed in the cotransfected cells. Only 30% of the initial radioactivity disappears, even by 10 min. This disappearance is completely insensitive to DIDS and is identical in magnitude and kinetics to the DIDS-insensitive fraction of the cotransfected cells, therefore providing evidence that it represents nonspecific diffusion. Unlabeled taurocholate was used in this experiment to optimize the chase effect, but similar results have been observed without unlabeled taurocholate (data not shown). Taken together, these studies show that CBATP can mediate specific, facilitated efflux of taurocholate in cotransfected COS cells and that its efflux properties can be detected more easily, over a longer duration of time and in the absence of the pharmacologic agent valinomycin originally used to promote nonspecific uptake of taurocholate by clamping the membrane potential. Now we could use this assay to examine the possibility that bile acid efflux mediated by CBATP is driven by ATP and/or membrane potential differences. COS cells were cotransfected with IBAT and CBATP and then, 48 h later, the cotransfected cells were incubated in 2-deoxyglucose and sodium azide under conditions associated with depletion of cellular ATP but without affecting cell viability. During this same 20-min interval, 400 μm[3H]taurocholate were also added to the medium for uptake studies. The cells were then washed extensively, and separate monolayers were incubated in the absence or presence of 1 mm DIDS and in the absence or presence of exogenous ATP in several different concentrations for an efflux assay of 5 min (Fig.3). The results show that there is no efflux in the absence of ATP. It also shows that efflux is absolutely dependent on ATP. The effect of extracellular ATP is concentration-dependent and saturable with aK m ≅ 10 μm andV max of 300 pmol/mg protein/min. To determine whether the electrochemical potential of the membrane drives bile acid efflux activity mediated by CBATP, we examined the effect of clamping the membrane potential with valinomycin. Cells were cotransfected with IBAT and CBATP and then, 48 h later, incubated with 150 μm [3H]taurocholate in the absence and presence of DIDS and in the absence or presence of 100 μm valinomycin, an ionophore that clamps the membrane potential (8Sippel C.J. Suchy F.J. Ananthanarayanan M. Perlmutter D.H. J. Biol. Chem. 1993; 268: 2083-2091Abstract Full Text PDF PubMed Google Scholar, 10Sippel C.J. McCollum M.J. Perlmutter D.H. J. Biol. Chem. 1994; 269: 2820-2826Abstract Full Text PDF PubMed Google Scholar, 11Cheung P.H. Luo W. Qiu Y. Zhang X. Earley K. Millirans P. Lin S.-H. J. Biol. Chem. 1993; 268: 24303-24310Abstract Full Text PDF PubMed Google Scholar). Cells were then washed and incubated for an additional 5 min at 22 °C in buffer supplemented with 5 mm ATP. The results show that valinomycin does not inhibit bile acid efflux (data not shown). Taken together, these data indicate that bile acid efflux mediated by CBATP is dependent on ATP but not on the electrochemical membrane potential. Thus, it cannot account for electrogenic bile acid transport demonstrated previously in canalicular membrane vesicles (26Nishida T. Chez M. Gaitman Z. Arias I.M. Hepatology. 1992; 16: 149ACrossref PubMed
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