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Organic Anion Transporting Polypeptide Mediates Organic Anion/HCO3− Exchange

1997; Elsevier BV; Volume: 272; Issue: 42 Linguagem: Inglês

10.1074/jbc.272.42.26340

1083-351X

Lisa M. Satlin, Vipul Amin, Allan W. Wolkoff,

Aldose Reductase and Taurine

Organic anion transporting polypeptide (oatp) is an integral membrane protein cloned from rat liver that mediates Na+-independent transport of organic anions such as sulfobromophthalein and taurocholic acid. Previous studies in rat hepatocytes suggested that organic anion uptake is associated with base exchange. To better characterize the mechanism of oatp-mediated organic anion uptake, we examined transport of taurocholate in a HeLa cell line stably transfected with oatp under the regulation of a zinc-inducible promoter (Shi, X., Bai, S., Ford, A. C., Burk, R. D., Jacquemin, E., Hagenbuch, B., Meier, P. J., and Wolkoff, A. W. (1995) J. Biol. Chem. 270, 25591–25595). Whereas noninduced transfected cells showed virtually no uptake of [3H]taurocholate, taurocholate uptake by induced cells was Na+-independent and saturable (K m = 19.4 ± 3.3 μm; V max = 62.2 ± 1.4 pmol/min/mg protein; n = 3). To test whether organic anion transport is coupled to HCO3− extrusion, we compared the rates of taurocholate-dependent HCO3− efflux from alkali-loaded noninduced and induced cells. Monolayers grown on glass coverslips were loaded with the pH-sensitive dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein; intracellular pH (pHi) was measured by excitation ratio fluorometry. Noninduced and induced cells were alkalinized to an equivalent pHi (∼7.7) by transient exposure to a 50 mmHCO3−, Cl−-free solution. In the absence of extracellular Cl− and taurocholate, isohydric reduction of superfusate HCO3−concentration from 50 to 25 mm resulted in an insignificant change in pHi over time (dpHi/dt) in both groups. Addition of 25 μm taurocholate to the superfusate led to a rapid fall in pHi in induced (−0.037 ± 0.011 pH units/min to pHi of 7.41 ± 0.14) but not in noninduced (0.003 ± 0.006 pH units/min to pHi of 7.61 ± 0.08) cells (p < 0.03). These data indicate that oatp-mediated taurocholate transport is Na+-independent, saturable, and accompanied by HCO3−exchange. We conclude that organic anion/base exchange is an important, potentially regulatable component of oatp function. Organic anion transporting polypeptide (oatp) is an integral membrane protein cloned from rat liver that mediates Na+-independent transport of organic anions such as sulfobromophthalein and taurocholic acid. Previous studies in rat hepatocytes suggested that organic anion uptake is associated with base exchange. To better characterize the mechanism of oatp-mediated organic anion uptake, we examined transport of taurocholate in a HeLa cell line stably transfected with oatp under the regulation of a zinc-inducible promoter (Shi, X., Bai, S., Ford, A. C., Burk, R. D., Jacquemin, E., Hagenbuch, B., Meier, P. J., and Wolkoff, A. W. (1995) J. Biol. Chem. 270, 25591–25595). Whereas noninduced transfected cells showed virtually no uptake of [3H]taurocholate, taurocholate uptake by induced cells was Na+-independent and saturable (K m = 19.4 ± 3.3 μm; V max = 62.2 ± 1.4 pmol/min/mg protein; n = 3). To test whether organic anion transport is coupled to HCO3− extrusion, we compared the rates of taurocholate-dependent HCO3− efflux from alkali-loaded noninduced and induced cells. Monolayers grown on glass coverslips were loaded with the pH-sensitive dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein; intracellular pH (pHi) was measured by excitation ratio fluorometry. Noninduced and induced cells were alkalinized to an equivalent pHi (∼7.7) by transient exposure to a 50 mmHCO3−, Cl−-free solution. In the absence of extracellular Cl− and taurocholate, isohydric reduction of superfusate HCO3−concentration from 50 to 25 mm resulted in an insignificant change in pHi over time (dpHi/dt) in both groups. Addition of 25 μm taurocholate to the superfusate led to a rapid fall in pHi in induced (−0.037 ± 0.011 pH units/min to pHi of 7.41 ± 0.14) but not in noninduced (0.003 ± 0.006 pH units/min to pHi of 7.61 ± 0.08) cells (p < 0.03). These data indicate that oatp-mediated taurocholate transport is Na+-independent, saturable, and accompanied by HCO3−exchange. We conclude that organic anion/base exchange is an important, potentially regulatable component of oatp function. The liver is the primary organ responsible for removal of organic anions such as bilirubin and sulfobromophthalein (BSP) 1The abbreviations used are: BSP, sulfobromophthalein; oatp, organic anion transporting polypeptide. from the circulation. These compounds are bound tightly to albumin, from which they are rapidly extracted by hepatocytes (1Scharschmidt B.F. Waggoner J.G. Berk P.D. J. Clin. Invest. 1975; 56: 1280-1292Crossref PubMed Scopus (204) Google Scholar, 2Stollman Y.R. Gartner U. Theilmann L. Ohmi N. Wolkoff A.W. J. Clin. Invest. 1983; 72: 718-723Crossref PubMed Scopus (75) Google Scholar). Functional studies in short term cultured rat hepatocytes (3Min A.D. Johansen K.L. Campbell C.G. Wolkoff A.W. J. Clin. Invest. 1991; 87: 1496-1502Crossref PubMed Scopus (49) Google Scholar, 4Wolkoff A.W. Samuelson A.C. Johansen K.L. Nakata R. Withers D.M. Sosiak A. J. Clin. Invest. 1987; 79: 1259-1268Crossref PubMed Scopus (85) Google Scholar) and isolated perfused rat liver (1Scharschmidt B.F. Waggoner J.G. Berk P.D. J. Clin. Invest. 1975; 56: 1280-1292Crossref PubMed Scopus (204) Google Scholar, 2Stollman Y.R. Gartner U. Theilmann L. Ohmi N. Wolkoff A.W. J. Clin. Invest. 1983; 72: 718-723Crossref PubMed Scopus (75) Google Scholar) revealed carrier-mediated kinetics for this uptake process. Recently, using a functional expression cloning strategy, a rat liver cDNA encoding a Na+-independent organic anion transport protein, oatp, was isolated (5Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. J. Clin. Invest. 1991; 88: 2146-2149Crossref PubMed Scopus (63) Google Scholar, 6Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (550) Google Scholar). Stable transfection of this cDNA into HeLa cells using a vector containing a zinc-inducible promoter has allowed for characterization of transporter activity in the absence of other hepatocyte cell membrane transporters that may mediate and/or modify organic anion uptake (7Shi X. Bai S. Ford A.C. Burk R.D. Jacquemin E. Hagenbuch B. Meier P.J. Wolkoff A.W. J. Biol. Chem. 1995; 270: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Whereas parent and noninduced transfected HeLa cells show no significant base line BSP transport, zinc-induced transfected cells avidly take up the organic anion in a manner identical to that previously described in cultured rat hepatocytes (7Shi X. Bai S. Ford A.C. Burk R.D. Jacquemin E. Hagenbuch B. Meier P.J. Wolkoff A.W. J. Biol. Chem. 1995; 270: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The mechanism by which oatp mediates organic anion transport has not been established. Studies in short term cultured rat hepatocytes showed that BSP uptake is stimulated by an outwardly directed pH gradient (pHi > pHo) (3Min A.D. Johansen K.L. Campbell C.G. Wolkoff A.W. J. Clin. Invest. 1991; 87: 1496-1502Crossref PubMed Scopus (49) Google Scholar), suggesting that a component of BSP uptake is associated with H+ cotransport or OH− (or HCO3−) exchange. The purpose of the present study was to determine whether oatp-mediated organic anion transport is coupled to base exchange. To this end, we used a fluorescence assay to examine the capacity of organic anions to stimulate HCO3− extrusion in alkali-loaded HeLa cells transfected with oatp cDNA under regulation of a zinc-inducible promoter. Whereas previous analyses of oatp-mediated organic anion uptake were performed with [35S]BSP (7Shi X. Bai S. Ford A.C. Burk R.D. Jacquemin E. Hagenbuch B. Meier P.J. Wolkoff A.W. J. Biol. Chem. 1995; 270: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), we found that the intense purple color of this compound in alkaline solutions limited visibility in the fluorescence functional assays described below. We thus chose to use the colorless organic anion taurocholate, also a substrate for oatp (8Kanai N. Lu R. Bao Y. Wolkoff A.W. Schuster V.L. Am. J. Physiol. 1996; 270: F319-F325PubMed Google Scholar), throughout this investigation. Initial studies were performed to validate that the characteristics of taurocholate transport mediated by oatp in these cells were similar to those previously described for BSP (7Shi X. Bai S. Ford A.C. Burk R.D. Jacquemin E. Hagenbuch B. Meier P.J. Wolkoff A.W. J. Biol. Chem. 1995; 270: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). HeLa cells (ATCC) that had been stably transfected with a plasmid in which oatp expression was regulated by a zinc-inducible promoter were maintained as described previously (7Shi X. Bai S. Ford A.C. Burk R.D. Jacquemin E. Hagenbuch B. Meier P.J. Wolkoff A.W. J. Biol. Chem. 1995; 270: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). For induction of expression, 100 μmZnSO4 was added to the culture medium for 24 h, and an additional 50 μm ZnSO4 was added for the final 18–20 h before use (7Shi X. Bai S. Ford A.C. Burk R.D. Jacquemin E. Hagenbuch B. Meier P.J. Wolkoff A.W. J. Biol. Chem. 1995; 270: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). For fluorescence assays, cells were plated on 2 × 5 mm pieces of No. 1 glass coverslips (Corning Glass, Corning, NY) and grown to confluence. Uptake of 22,34-[3H]-sodium taurocholate (specific activity 58 Ci/mmol; gift from Dr. Alan Hofmann) by HeLa cells was determined in the absence of bovine serum albumin as described previously for studies of BSP transport (7Shi X. Bai S. Ford A.C. Burk R.D. Jacquemin E. Hagenbuch B. Meier P.J. Wolkoff A.W. J. Biol. Chem. 1995; 270: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) utilizing modified serum-free medium containing (in mm): 135 NaCl, 1.2 MgCl2, 0.81 MgSO4, 27.8 glucose, 2.5 CaCl2, and 25 HEPES adjusted to pH 7.2 with NaOH. Cell protein was determined in replicate plates by the method of Lowry et al. (9Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as standard. These studies were performed at taurocholate concentrations of 1–200 μm. Preliminary studies showed that uptake of [3H]taurocholate by zinc-induced cells was linear for at least 5 min. Uptake of [3H]taurocholate was determined at 4 and 37 °C over the initial 5 min of linear uptake. Data were analyzed by nonlinear least squares regression (SigmaPlot version 5.0, Jandel Scientific, San Rafael, CA), and K m andV max were calculated. Medium was prepared in which NaCl was substituted isosmotically by KCl. Initial uptake of [3H]taurocholate was determined over 5 min in KCl-substituted medium. Modified serum-free medium was prepared as above except that the pH was adjusted to 6.0 or 8.0 with 1m NaOH. Cells were washed twice with 1.5 ml of either pH 6.0 or 8.0 medium in which they were then incubated for 30 min at 37 °C. Unidirectional pH gradients were generated by rapidly incubating the cells in medium of the opposite pH, as described previously (3Min A.D. Johansen K.L. Campbell C.G. Wolkoff A.W. J. Clin. Invest. 1991; 87: 1496-1502Crossref PubMed Scopus (49) Google Scholar). Initial uptake of [3H]taurocholate was determined over 5 min after addition of the organic anion to the medium. pHi was estimated by excitation ratio fluorometry using a silicon-intensified target video camera (Dage, Michigan City, IN) attached to the cine port of a Nikon Diaphot inverted microscope, as described previously (10Satlin L.M. Matsumoto T. Schwartz G.J. Am. J. Physiol. 1992; 262: F199-F208Crossref PubMed Google Scholar, 11Silver R.B. Mennitt P.A. Satlin L.M. Am. J. Physiol. 1996; 270: F539-F547PubMed Google Scholar). Cells were identified using a 50× Leitz water objective. The green fluorescence of 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF; Molecular Probes) was visualized with a Nikon B filter cassette. There was no detectable autofluorescence of the cells or any of the solutions used. Sequential fluorescence images of BCECF-loaded cells were obtained at 10-s intervals at excitation wavelengths of 490 and 440 nm (emission at 530 nm) and stored on a Digital Instruments computer. Data were analyzed with a commercially available digital video image analysis system (MetaFluor; Universal Imaging, West Chester, PA). An intracellular calibration was performed at the conclusion of each experiment using the nigericin technique, as described previously (10Satlin L.M. Matsumoto T. Schwartz G.J. Am. J. Physiol. 1992; 262: F199-F208Crossref PubMed Google Scholar,11Silver R.B. Mennitt P.A. Satlin L.M. Am. J. Physiol. 1996; 270: F539-F547PubMed Google Scholar). High-K+ intracellular calibration buffers (TableI, solution 4) containing 10 μm nigericin (Molecular Probes) were adjusted to pH values of 6.8, 7.3, and 7.8.Table IComposition of solutionsSolution1234NaCl11545.0Na gluconate11590NaHCO3252550KCl99.5K2HPO42.52.52.5KH2PO40.5CaCl22.00.2Ca acetate4.04.0MgSO41.21.21.21.6MgCl20.3NaH2PO413.3Na2HPO42.5Na lactate4.04.04.0Na3citrate1.01.01.0l-Alanine6.06.06.0Glucose5.55.55.5O2 (%)959590CO2 (%)5510pH7.47.47.4VariableAll solute concentrations are in millimolar. Solutions were adjusted to 290 mosmol/kg H2O by addition of H2O or the major salt and were gassed at 37 °C. Open table in a new tab All solute concentrations are in millimolar. Solutions were adjusted to 290 mosmol/kg H2O by addition of H2O or the major salt and were gassed at 37 °C. Coverslips to which cell monolayers were attached were placed on the floor of a temperature-controlled specimen chamber, bathed with standard perfusate (Table I, solution 1) at 37 °C, and gassed with 95% O2, 5% CO2. Bath exchanges were performed manually by rapidly replacing the ∼1.5 ml volume of bathing solution three times. After equilibration, cell monolayers were exposed to 20 μm BCECF acetoxymethyl ester for 10–15 min. After rinsing and obtaining base line pHi measurements, cells were alkali-loaded by isohydric application of a 50 mmHCO3−, Cl−-free solution (Table I, solution 3). This solution was prepared by replacing 25 mm sodium gluconate in the standard Cl−-free solution (Table I, solution 2) with an additional 25 mmNaHCO3 and gassing with 90% O2, 10% CO2. After obtaining a stable 490 nm/450 nm fluorescence signal over a given cell, cells were alkali-loaded (10Satlin L.M. Matsumoto T. Schwartz G.J. Am. J. Physiol. 1992; 262: F199-F208Crossref PubMed Google Scholar, 12Ganz M.B. Boyarsky G. Sterzel R.B. Boron W.F. Nature. 1989; 337: 648-651Crossref PubMed Scopus (220) Google Scholar) by abruptly reducing the HCO3− concentration in the superfusate to 25 mm and CO2 to 5% at a constant pH of 7.4 (Table I, solution 2). In the continued absence of Cl−, the 490 nm/450 nm fluorescence ratios were monitored. Thereafter, taurocholate (2.5 or 25 μm) and finally Cl− (Table I, solution 1) were added to the superfusate and 490 nm/450 nm emissions monitored. For each cell studied, the initial rate of change in pHi(dpHi/dt) observed in response to a change in superfusate was calculated by linear regression analysis, as described previously (10Satlin L.M. Matsumoto T. Schwartz G.J. Am. J. Physiol. 1992; 262: F199-F208Crossref PubMed Google Scholar, 11Silver R.B. Mennitt P.A. Satlin L.M. Am. J. Physiol. 1996; 270: F539-F547PubMed Google Scholar). At least 5 data points were used for each slope determination. As buffer capacity measurements of HeLa cells were not performed, cellular OH−(HCO3−) fluxes were not determined. Results are expressed as mean ± S.E.; n represents the number of monolayers. In fluorescence studies, data from multiple (2Stollman Y.R. Gartner U. Theilmann L. Ohmi N. Wolkoff A.W. J. Clin. Invest. 1983; 72: 718-723Crossref PubMed Scopus (75) Google Scholar, 3Min A.D. Johansen K.L. Campbell C.G. Wolkoff A.W. J. Clin. Invest. 1991; 87: 1496-1502Crossref PubMed Scopus (49) Google Scholar, 4Wolkoff A.W. Samuelson A.C. Johansen K.L. Nakata R. Withers D.M. Sosiak A. J. Clin. Invest. 1987; 79: 1259-1268Crossref PubMed Scopus (85) Google Scholar, 5Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. J. Clin. Invest. 1991; 88: 2146-2149Crossref PubMed Scopus (63) Google Scholar) cells per plate were averaged to provide a mean value. Significant differences between paired data were determined by paired t test. Comparisons of unpaired data were performed by t test or analysis of variance and multiple range test, as appropriate. The software programs SigmaPlot (version 5.0) and SigmaStat (Jandel Scientific) were used for all statistical analyses. Significance was asserted if p < 0.05. Taurocholate uptake by induced oatp-transfected HeLa cells was rapid and linear over 5 min at 37 °C (Fig.1). In contrast, little ligand associated with induced cells at 4 °C (Figs. 1 and 2). The initial rate of taurocholate uptake in induced cells exceeded the rates of nonspecific association of the ligand with noninduced cells measured at 37 °C and both induced and noninduced cells studied at 4 °C (Fig.2).Figure 2Uptake of [3H]taurocholate by zinc-induced and noninduced oatp-transfected HeLa cells. The initial rate of uptake over 5 min of 1 μm taurocholate by induced cells significantly exceeded that measured in noninduced cells at 37 °C. Uptake by noninduced cells at 37 °C did not differ from the negligible nonspecific taurocholate uptake measured in both induced and noninduced cells at 4 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Initial uptake of taurocholate was saturable (Fig.3). The mean K m was 19.4 ± 3.3 μm and V max was 62.2 ± 1.4 pmol/min/mg protein (n = 3 experiments). As previously reported for BSP uptake in rat hepatocytes (3Min A.D. Johansen K.L. Campbell C.G. Wolkoff A.W. J. Clin. Invest. 1991; 87: 1496-1502Crossref PubMed Scopus (49) Google Scholar), substitution of extracellular Na+ by K+ had no effect on the initial rate of [3H]taurocholate uptake (102% of control; n = 2), confirming that oatp-mediated taurocholate uptake is Na+-independent. These studies also suggest that this process is not electrogenic, as exposure to a high K+ bathing solution that should result in cell depolarization did not alter the rate of taurocholate uptake. Previous studies in cultured rat hepatocytes identified a pH dependence of BSP uptake (3Min A.D. Johansen K.L. Campbell C.G. Wolkoff A.W. J. Clin. Invest. 1991; 87: 1496-1502Crossref PubMed Scopus (49) Google Scholar). To examine the effect of pH on oatp-mediated taurocholate transport, unidirectional pH gradients were established and the initial rate of taurocholate uptake determined. With an outwardly directed OH− gradient (pHi/pHo = 8/6), in the nominal absence of HCO3−/CO2, taurocholate uptake was >2-fold higher than uptake observed in the presence of an inwardly directed pH gradient (pHi/pHo = 6/8) (Fig. 4). These results suggest that the pH optimum for oatp-mediated taurocholate transport is ∼7.2 and that uptake is associated with OH− (or HCO3−) exchange or H+cotransport. In preliminary experiments, we determined that extracellular Cl−replacement with gluconate led to a reversible 0.19 ± 0.04 pH unit increase in steady-state pHi of parent HeLa cells (7.33 ± 0.10 to 7.52 ± 0.09; n = 3;p < 0.05). Pretreatment of cells with 0.5 mm 4–4′-diisothiocyanostilbene-2,2′-disulfonic acid inhibited this cell alkalinization response (ΔpHi = 0.05 ± 0.04 pH unit; n = 3), consistent with the presence of Cl−/HCO3− exchange, a transport pathway previously proposed to be present in HeLa cells (14Holsey C. Cragoe Jr., E.J. Nair C.N. J. Cell Physiol. 1990; 142: 586-591Crossref PubMed Scopus (7) Google Scholar). We exploited the activity and reversibility of this Cl−/HCO3− exchanger to load HeLa cells with HCO3−. Exposure to a 50 mm HCO3−, Cl−-free solution led to a significant increase in pHi in noninduced cells (7.42 ± 0.07 to 7.64 ± 0.07; n = 8; p < 0.05) over ∼10 min. Induced cells, characterized by a resting pHi of 7.59 ± 0.14, showed insignificant additional alkalinization (to pHi 7.76 ± 0.10; n = 7;p = 0.3) in response to this maneuver. Reduction of the extracellular HCO3− concentration from 50 to 25 mm in the continued absence of Cl− led to no significant change in pHi in either noninduced or induced cells (Figs. 5 and 7).Figure 7Effect of 25 μm taurocholate on steady state pHi values of noninduced (A) and zinc-induced (B) oatp-transfected HeLa cells during recovery from an acute alkali load. The pHi values indicated include resting in standard perfusate, maximal after alkali loading, steady state on reduction of extracellular HCO3− from 50 to 25 mm in the absence of Cl−, steady state following addition of 25 μm taurocholate to the bathing medium, and value 5 min after restoration of Cl− to the superfusate. In noninduced cells (A; n = 7), pHi recovery was observed only after restoration of Cl− to the bath. In contrast, in induced cells (B; n = 6), recovery of pHi was observed after exposure to taurocholate. Results are means ± S.E. *, p < 0.05 or +, 0.05 < p < 0.10 compared with pHi in 25 mm HCO3−, Cl−- and taurocholate-free medium.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fig. 5 A depicts a representative tracing of taurocholate-dependent pHi recovery of a single alkali-loaded noninduced HeLa cell. In this experiment, the cell was alkali-loaded from a resting pHi of 7.4 to a maximum pHi of 7.6. pHi recovery (i.e.extrusion of the HCO3− load) was followed initially in the absence of Cl− and taurocholate and then following the sequential addition of 2.5 and 25 μmtaurocholate to the bathing medium. Note that pHi did not return toward base line until Cl− was restored to the superfusate. We found that the rate of Cl−-independent pHirecovery in noninduced cells did not differ from zero on addition of 2.5 or 25 μm taurocholate to the bathing medium (Fig.6). The absence of significant taurocholate-dependent pHi recovery in noninduced cells was reflected in the absence of change in pHi observed in response to exposure to either 2.5 μm (7.56 ± 0.06 to 7.59 ± 0.06) or 25 μm (Fig. 7 A) taurocholate. Restoration of extracellular Cl− to the bathing medium led to significant (p < 0.05) pHi recovery in cells exposed to 2.5 μm(−0.041 ± 0.009 pH units/min to a final pHi of 7.35 ± 0.08) and 25 μm (−0.043 ± 0.011 pH units/min to a final pHi of 7.31 ± 0.09) taurocholate. A representative tracing of taurocholate-dependent pHi recovery of a single alkali-loaded zinc-induced HeLa cell is shown in Fig. 5 B. In this experiment, the cell was alkali-loaded from a resting pHi of 7.5 to a maximum pHi of 7.8. In the absence of Cl− and taurocholate, pHi remained high on reduction of the extracellular HCO3−concentration from 50 to 25 mm. Addition of 2.5 μm taurocholate, however, led to a prompt, albeit partial, reduction in pHi. On exposure to 25 μm taurocholate, pHi rapidly fell to base line. Readdition of Cl− to the extracellular medium had no further effect on pHi. The rate of Cl−-independent pHi recovery in induced cells was −0.024 ± 0.010 pH units/min in the presence of 2.5 μm taurocholate (Fig. 6); pHi fell from its maximum of 7.81 ± 0.08 to 7.69 ± 0.07 (p = 0.005) in this group of cells. Restoration of Cl− to the medium bathing these cells led to a further reduction in pHi at a rate of −0.037 ± 0.007 pH units/min to stabilize at a pHi of 7.40 ± 0.04 (p < 0.05 compared with pHi in absence of Cl−). In a separate group of induced cells, addition of 25 μmtaurocholate caused cell pHi to fall at a rate of −0.037 ± 0.011 pH units/min (Fig. 6) from a maximal pHi of 7.69 ± 0.11 to 7.41 ± 0.14 (Fig.7 B). In this group of cells, restoration of cell Cl− led to no further decrease in pHi, which had already reached base line (dpHi/dt = −0.014 ± 0.015 pH unit/min to 7.37 ± 0.16; p = NS compared with pHi in 25 μm taurocholate, Cl−-free medium and initial resting pHi). Cumulative evidence suggests that a variety of cells possess a zinc-sensitive H+ conductance (13Lukacs G.L. Kapus A. Nanda A. Romanek R. Grinstein S. Am. J. Physiol. 1993; 265: C3-C14Crossref PubMed Google Scholar). To determine whether exposure to ZnSO4 altered H+/HCO3− transport in HeLa cells, we compared resting pHi and Cl−/HCO3− exchange activity in parent HeLa cells grown in the absence or presence of 100 μm ZnSO4 for 48 h. Our observation of statistically similar resting pHi values (7.16 ± 0.08 versus 7.27 ± 0.07; p = 0.36) and increases in pHi (0.21 ± 0.08 versus0.26 ± 0.10; p = 0.74) in response to extracellular Cl− replacement with gluconate in cells grown in the presence (n = 5) and absence (n = 5), respectively, of ZnSO4 suggested it unlikely that this compound systematically altered pHiregulatory pathways or capacity for Cl−/HCO3− exchange. Functional characterization of individual transport pathways mediating uptake of organic anions in the rat liver has been complicated by the presence in hepatocytes of multiple endogenous organic anion transport proteins (3Min A.D. Johansen K.L. Campbell C.G. Wolkoff A.W. J. Clin. Invest. 1991; 87: 1496-1502Crossref PubMed Scopus (49) Google Scholar, 15Wolkoff A.W. Arias I.M. Boyer J.L. Fausto N. Jakoby W.B. Schachter D. Shafritz D.A. The Liver: Biology and Pathobiology. 3rd Ed. Raven Press, Ltd., New York1994: 179-188Google Scholar). To selectively characterize the mechanism of oatp-mediated taurocholate transport, we used HeLa cells permanently transfected with a eukaryotic expression vector containing an inducible oatp cDNA (7Shi X. Bai S. Ford A.C. Burk R.D. Jacquemin E. Hagenbuch B. Meier P.J. Wolkoff A.W. J. Biol. Chem. 1995; 270: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The results of the present study indicate that oatp-mediated transport of taurocholate is Na+-independent, saturable, and temperature-dependent. These transport characteristics are similar to those previously described for oatp-mediated BSP transport (7Shi X. Bai S. Ford A.C. Burk R.D. Jacquemin E. Hagenbuch B. Meier P.J. Wolkoff A.W. J. Biol. Chem. 1995; 270: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Similarly, we observed that taurocholate uptake is stimulated by imposition of an outwardly directed pH gradient, as previously reported for BSP transport in cultured hepatocytes (3Min A.D. Johansen K.L. Campbell C.G. Wolkoff A.W. J. Clin. Invest. 1991; 87: 1496-1502Crossref PubMed Scopus (49) Google Scholar). Coupling of organic anion transport to pH gradients could arise from anion/OH− (or HCO3−) exchange, a process equivalent to anion-H+ cotransport. Indeed, cholate uptake in the intact liver has been postulated to be mediated, at least in part, by bile acid/OH− (or HCO3−) exchange (16Blitzer B.L. Terzakis C. Scott K.A. J. Biol. Chem. 1986; 261: 12042-12046Abstract Full Text PDF PubMed Google Scholar, 17Veith C.M. Thalhammer T. Felberbauer F.X. Graf J. Biochim. Biophys. Acta. 1992; 1103: 51-61Crossref PubMed Scopus (15) Google Scholar). To test the hypothesis that oatp-mediated taurocholate uptake is coupled to base exchange, we examined the capacity of extracellular taurocholate to stimulate base efflux in HCO3−-loaded noninduced and induced HeLa cells. As shown in Figs. 5 and 6, extracellular taurocholate stimulated HCO3−extrusion in alkali-loaded induced cells alone. These results indicate that oatp expression confers on HeLa cells the capacity for taurocholate/HCO3− exchange. The recovery of pHi in induced cells to a value below their initial resting pHi may represent “overshoot” of taurocholate uptake. The cessation of taurocholate/HCO3− exchange upon reduction of pHi may explain, at least in part, the reduction in organic anion uptake previously observed in cells depleted of ATP (4Wolkoff A.W. Samuelson A.C. Johansen K.L. Nakata R. Withers D.M. Sosiak A. J. Clin. Invest. 1987; 79: 1259-1268Crossref PubMed Scopus (85) Google Scholar,7Shi X. Bai S. Ford A.C. Burk R.D. Jacquemin E. Hagenbuch B. Meier P.J. Wolkoff A.W. J. Biol. Chem. 1995; 270: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Although not examined in the present study, pHi would be expected to fall with ATP depletion, reducing the driving force for organic anion/HCO3− exchange. Our results indicate that oatp mediates Na+-independent, high affinity transport of taurocholate. A Na+-dependent bile acid transporter (ntcp) cloned from rat liver (18Hagenbuch B. Stieger B. Foguet M. Lubbert H. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10629-10633Crossref PubMed Scopus (455) Google Scholar) has also been recently characterized. COS-7 cells transfected with ntcp cDNA revealed Na+-dependent taurocholate transport with aK m of 29 μm (19Boyer J.L. Ng O.C. Ananthanarayanan M. Hofmann A.F. Schteingart C.D. Hagenbuch B. Stieger B. Meier P.J. Am. J. Physiol. 1994; 266: G382-G387Crossref PubMed Google Scholar). Although under normal conditions the majority of bile acid uptake by the liver is thought to be Na+-dependent (20Meier P.J. Am. J. Physiol. 1995; 269: G801-G812PubMed Google Scholar), the relative contributions of oatp and ntcp to conjugated bile acid transport in various pathobiologic states remain to be determined. Immunocytochemical studies indicate that oatp is localized to the basolateral (sinusoidal) plasma membrane of hepatocytes, a location consistent with its presumed role in clearing ligands from the circulation (21Bergwerk A.J. Shi X. Ford A.C. Kanai N. Jacquemin E. Burk R.D. Bai S. Novikoff P.M. Steiger B. Meier P.J. Schuster V.L. Wolkoff A.W. Am. J. Physiol. 1996; 271: G231-G238PubMed Google Scholar). As such, the transporter would be expected to extrude cellular HCO3−, if that were the physiologic substrate extruded in exchange for organic anion uptake, into the circulation. In rat liver, both a Na+/H+antiporter (22Moseley R.H. Meier P.J. Aronson P.S. Boyer J.L. Am. J. Physiol. 1986; 250: G35-G43PubMed Google Scholar) and Na+-HCO3−symporter (23Gleeson D. Smith N.D. Boyer J.L. J. Clin. Invest. 1989; 84: 312-321Crossref PubMed Scopus (95) Google Scholar, 24Renner E.L. Lake J.R. Scharschmidt B.F. Zimmerli B. Meier P.J. J. Clin. Invest. 1989; 83: 1225-1235Crossref PubMed Scopus (56) Google Scholar) are localized to the basolateral membrane; the latter transporter is believed to function as a base loader under resting conditions (25Fitz J.G. Lidofsky S.D. Weisiger R.A. Xie M.H. Cochran M. Grotmol T. Scharschmidt B.F. J. Membr. Biol. 1991; 122: 1-10Crossref PubMed Scopus (15) Google Scholar). Thus, the presence of a basolateral organic anion/base exchanger might provide a pathway for OH−(or HCO3−) exit from the hepatocyte. Although the pHi of the hepatocyte is ∼7.2 (26Benedetti A. Strazzabosco M. Corasanti J.G. Haddad P. Graf J. Boyer J.L. Am. J. Physiol. 1991; 261: G512-G522PubMed Google Scholar) and thus presumably slightly more acidic than the portal blood (pH 7.4), the outward movement of base may be favored by local outwardly directed gradients established by the basolateral Na+-HCO3− symporter and the negative cell potential (−40 mV) (27Fitz J.G. Scharschmidt B.F. Am. J. Physiol. 1987; 252: G56-G64PubMed Google Scholar, 28Graf J. Henderson R.M. Krumpholz B. Boyer J.L. J. Membrane Biol. 1987; 95: 241-254Crossref PubMed Scopus (70) Google Scholar). It should be noted that our demonstration of taurocholate/HCO3− exchange does not prove that HCO3− is the physiologic substrate for oatp in vivo. Other potential substrates capable of driving uphill oatp-mediated organic anion transport include α-ketoglutarate and metabolic intermediates that accumulate to relatively high concentrations within the hepatocyte. While beyond the scope of the present study, identification of the physiologic substrate(s) for oatp remains an important goal for future investigation.