INTRODUCTION

The liver is the primary organ responsible for removal of organic anions such as bilirubin and sulfobromophthalein (BSP)¹ from the circulation. These compounds are bound tightly to albumin, from which they are rapidly extracted by hepatocytes (1, 2). Functional studies in short term cultured rat hepatocytes (3, 4) and isolated perfused rat liver (1, 2) 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 (5, 6). 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 (7). 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 (7).

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 (pH_i > pH_o) (3), 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 (7), 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 (8), 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 (7).

EXPERIMENTAL PROCEDURES

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 (7). For induction of expression, 100 µM ZnSO₄ 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 (7). 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 (7) 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. (9) 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 and V_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 1 M 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 (3). Initial uptake of [³H]taurocholate was determined over 5 min after addition of the organic anion to the medium.

pH_i 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 (10, 11). 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 (10, 11). High-K⁺ intracellular calibration buffers (Table I, solution 4) containing 10 µM nigericin (Molecular Probes) were adjusted to pH values of 6.8, 7.3, and 7.8. 

Table I. Composition of solutions 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. Solution 1 2 3 4 NaCl 115 45.0 Na gluconate 115 90 NaHCO3 25 25 50 KCl 99.5 K2HPO4 2.5 2.5 2.5 KH2PO4 0.5 CaCl2 2.0 0.2 Ca acetate 4.0 4.0 MgSO4 1.2 1.2 1.2 1.6 MgCl2 0.3 NaH2PO4 13.3 Na2HPO4 2.5 Na lactate 4.0 4.0 4.0 Na3 citrate 1.0 1.0 1.0 L-Alanine 6.0 6.0 6.0 Glucose 5.5 5.5 5.5 O2 (%) 95 95 90 CO2 (%) 5 5 10 pH 7.4 7.4 7.4 Variable

Kinetic Analysis of Taurocholate/HCO₃ Exchange

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 pH_i measurements, cells were alkali-loaded by isohydric application of a 50 mM HCO₃, 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 mM NaHCO₃ 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 (10, 12) 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 pH_i (dpH_i/dt) observed in response to a change in superfusate was calculated by linear regression analysis, as described previously (10, 11). 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 (2-5) 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.

RESULTS

Characteristics of [³H]Taurocholate Uptake by HeLa Cells

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).

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 (3), 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.

Effect of pH Gradients on Initial Uptake of [³H]Taurocholate

Previous studies in cultured rat hepatocytes identified a pH dependence of BSP uptake (3). 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 (pH_i/pH_o = 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 (pH_i/pH_o = 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.

Kinetics of Taurocholate/HCO₃ Exchange Activity

HCO₃ Loading of HeLa Cells

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 pH_i 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 (pH_i = 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 (14).

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 pH_i 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 pH_i of 7.59 ± 0.14, showed insignificant additional alkalinization (to pH_i 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 pH_i in either noninduced or induced cells (Figs. 5 and 7).

Fig. 5A depicts a representative tracing of taurocholate-dependent pH_i recovery of a single alkali-loaded noninduced HeLa cell. In this experiment, the cell was alkali-loaded from a resting pH_i of 7.4 to a maximum pH_i of 7.6. pH_i 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 µM taurocholate to the bathing medium. Note that pH_i did not return toward base line until Cl was restored to the superfusate.

We found that the rate of Cl-independent pH_i recovery 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 pH_i recovery in noninduced cells was reflected in the absence of change in pH_i observed in response to exposure to either 2.5 µM (7.56 ± 0.06 to 7.59 ± 0.06) or 25 µM (Fig. 7A) taurocholate. Restoration of extracellular Cl to the bathing medium led to significant (p < 0.05) pH_i recovery in cells exposed to 2.5 µM (0.041 ± 0.009 pH units/min to a final pH_i of 7.35 ± 0.08) and 25 µM (0.043 ± 0.011 pH units/min to a final pH_i of 7.31 ± 0.09) taurocholate.

Effect of extracellular taurocholate on Cl-independent HCO

A representative tracing of taurocholate-dependent pH_i recovery of a single alkali-loaded zinc-induced HeLa cell is shown in Fig. 5B. In this experiment, the cell was alkali-loaded from a resting pH_i of 7.5 to a maximum pH_i of 7.8. In the absence of Cl and taurocholate, pH_i 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 pH_i. On exposure to 25 µM taurocholate, pH_i rapidly fell to base line. Readdition of Cl to the extracellular medium had no further effect on pH_i.

The rate of Cl-independent pH_i recovery in induced cells was 0.024 ± 0.010 pH units/min in the presence of 2.5 µM taurocholate (Fig. 6); pH_i 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 pH_i at a rate of 0.037 ± 0.007 pH units/min to stabilize at a pH_i of 7.40 ± 0.04 (p < 0.05 compared with pH_i in absence of Cl).

In a separate group of induced cells, addition of 25 µM taurocholate caused cell pH_i to fall at a rate of 0.037 ± 0.011 pH units/min (Fig. 6) from a maximal pH_i of 7.69 ± 0.11 to 7.41 ± 0.14 (Fig. 7B). In this group of cells, restoration of cell Cl led to no further decrease in pH_i, which had already reached base line (dpH_i/dt = 0.014 ± 0.015 pH unit/min to 7.37 ± 0.16; p = NS compared with pH_i in 25 µM taurocholate, Cl-free medium and initial resting pH_i).

Effect of ZnSO₄ on Resting pH_i and Cl/HCO₃ Exchange in Parent HeLa Cells

Cumulative evidence suggests that a variety of cells possess a zinc-sensitive H⁺ conductance (13). To determine whether exposure to ZnSO₄ altered H⁺/HCO₃ transport in HeLa cells, we compared resting pH_i 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 pH_i values (7.16 ± 0.08 versus 7.27 ± 0.07; p = 0.36) and increases in pH_i (0.21 ± 0.08 versus 0.26 ± 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 pH_i regulatory pathways or capacity for Cl/HCO₃ exchange.

DISCUSSION

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 (3, 15). 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 (7).

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 (7). 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 (3).

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 (16, 17). 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 pH_i in induced cells to a value below their initial resting pH_i may represent "overshoot" of taurocholate uptake. The cessation of taurocholate/HCO₃ exchange upon reduction of pH_i may explain, at least in part, the reduction in organic anion uptake previously observed in cells depleted of ATP (4, 7). Although not examined in the present study, pH_i 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 (18) has also been recently characterized. COS-7 cells transfected with ntcp cDNA revealed Na⁺-dependent taurocholate transport with a K_m of 29 µM (19). Although under normal conditions the majority of bile acid uptake by the liver is thought to be Na⁺-dependent (20), 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 (21). 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 (22) and Na⁺-HCO₃ symporter (23, 24) are localized to the basolateral membrane; the latter transporter is believed to function as a base loader under resting conditions (25). Thus, the presence of a basolateral organic anion/base exchanger might provide a pathway for OH (or HCO₃) exit from the hepatocyte. Although the pH_i of the hepatocyte is ~7.2 (26) 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) (27, 28).

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 -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.