Stoichiometry of organic anion/dicarboxylate exchange in membrane vesicles from rat renal cortex and hOAT1-expressing cells

Amy Aslamkhan,1 Yong-Hae Han,1 Ramsey Walden,1 Douglas H. Sweet,2 and John B. Pritchard1

1Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; and 2Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425

Submitted 8 April 2003 ; accepted in final form 26 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although membrane vesicle studies have established the driving forces that mediate renal organic anion secretion and the organic anion transporter Oat1 has now been cloned in several species, its stoichiometry has remained uncertain. In this study, we used electrophysiology, kinetic measurements, and static head experiments to determine the coupling ratio for Oat1-mediated organic anion/dicarboxylate exchange. Initial experiments demonstrated that uptake of PAH by voltage-clamped Xenopus laevis oocytes expressing rOat1 led to net entry of positive charge, suggesting that coupling was one-to-one. This conclusion was confirmed by kinetic analysis of PAH and glutarate fluxes in native basolateral membrane vesicles from the rat renal cortex, which showed a Hill coefficient of 1. Similarly, static head experiments on the rat vesicles also showed a 1:1 coupling ratio. To confirm these conclusions in a system expressing a single cloned transporter, Madin-Darby canine kidney cells were stably transfected with the human exchanger hOAT1. The hOAT1-expressing cell line showed extensive PAH transport, which was very similar in all respects to transport expressed by hOAT1 in Xenopus oocytes. Its Km for PAH was 8 µM and glutarate effectively trans-stimulated PAH transport. When stoichiometry was assessed using plasma membranes isolated from the hOAT1-expressing cells, both kinetic and static head data indicated that hOAT1 also demonstrated a 1:1 coupling between organic anion and dicarboxylate.

p-aminohippurate; hOAT1; organic anion transport; anion exchange; Madin-Darby canine kidney cells


A CRITICAL FUNCTION of the kidney is elimination of potentially toxic chemicals, both foreign and endogenous, from the body (17). The primary effector for the excretion of negatively charged chemicals and metabolites is the classical organic anion secretory system that transports PAH and other small anions (<500 Da). A good substrate such as PAH may be completely cleared from the renal plasma in a single pass through the kidney. In the 1980s, membrane vesicle studies led to the elucidation of the mechanisms and driving forces that enable this system to function so effectively (14, 16, 23). The critical uphill step in the secretory process was shown to be basolateral exchange of the anionic drug or xenobiotic for intracellular {alpha}-ketoglutarate ({alpha}-KG) (15). Two organic anion transporters, Oat1 and Oat3, have been localized to the basolateral membrane (BLM) (5, 7, 8, 25, 2729). Of these, Oat1 was first to be cloned and demonstrated to be an organic anion/{alpha}-KG exchanger, initially in rats (22, 28) and later in a number of other species, including humans (6, 8, 11, 19). Recently, we showed that rat Oat3 is also an organic anion/{alpha}-KG exchanger (24).

Surprisingly, given all of this attention, the stoichiometry of Oat1 has remained uncertain. The original experiments conducted using membrane vesicles isolated from rat kidney showed no change in PAH uptake when membrane potential was altered by changing potassium gradients in the presence of the potassium ionophore valinomycin (14). These data suggested that exchange was electroneutral, i.e., one divalent {alpha}-KG molecule being exchanged for two monovalent PAH molecules. Similar data were obtained in bovine vesicles (21), and a comparable conclusion was reached based on observations in the intact tubule (30). Even in Xenopus laevis oocytes expressing the cloned rat transporter rOat1, no effect on PAH transport was observed when the oocytes were depolarized (28). Because of the consistency of these findings from different laboratories, the stoichiometry was not determined directly. However, at least one study using rabbit renal BLM vesicles did show increased PAH uptake when membrane potential was inside negative and decreased uptake when it was inside positive (12). Furthermore, when Burckhardt et al. (3) examined the electrophysiology of organic anion exchange in oocytes expressing winter flounder Oat1 under voltage-clamped conditions, entry of organic anions was associated with net entry of positive charge or exit of negative charge. This result is most simply explained by a 1:1 exchange of organic anion for dicarboxylate. This issue, which has major implications in terms of transport mechanism at the molecular level, clearly requires resolution.

In the studies described below, we reexamined the stoichiometry of Oat1/OAT1 through 1) electrophysiological measurements in voltage-clamped rOat1-expressing Xenopus oocytes and 2) kinetic measurements and static head experiments in native BLMs isolated from rat kidney and in membranes isolated from Madin-Darby canine kidney (MDCK) cells stably expressing the human ortholog of the PAH/{alpha}-KG exchanger, hOAT1. In agreement with the electrophysiology data, the vesicle data show that the coupling ratio of PAH to {alpha}-KG is 1:1. Specifically, in both membrane preparations, the initial rate of PAH uptake was a hyperbolic function of the intravesicular concentration of counterion (Hill coefficient ~1) and simultaneous 10-fold gradients for PAH and dicarboxylate balanced one another, yielding no net flux in static head experiments.


    METHODS AND MATERIALS
 TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

[3H]PAH (1.28 Ci/mmol) and [3H]taurocholic acid (2.10 Ci/mmol) were purchased from New England Nuclear (Boston, MA). [14C]urate (55 mCi/mmol) and [14C]glutarate (15.6 mCi/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO) and ICN (Irvine, CA), respectively. Unlabeled PAH and quinine were obtained from Sigma (St. Louis, MO). All other chemicals were obtained from commercial sources and were of the highest grade available.

Animals

Rats. Kidneys were obtained from 300-g male Sprague-Dawley rats purchased from Taconic Farms (Germantown, NY) and maintained in the animal quarters at National Institute of Environmental Health Sciences (NIEHS) for 1 wk or less. For tissue harvest, animals were euthanized with 100% CO2 and the kidneys were immediately removed to ice-cold, oxygenated saline for preparation of isolated BLM.

Frogs. Female X. laevis were purchased from Xenopus One (Ann Arbor, MI). The animals were anesthetized with 0.3% tricaine (Sigma), decapitated, pithed, and the ovaries were removed. Stage V and VI oocytes were isolated by collagenase digestion as previously described (6, 13, 26). All animal procedures were carried out in accord with protocols approved by the NIEHS Animal Care and Use Committee.

Electrophysiology

Three days after cRNA injection, oocyte membrane currents were measured using a conventional two-electrode (3 M KCl; resistance of ~1 m{Omega}) voltage-clamp technique (Geneclamp 500B, Axon Instruments, Foster City, CA). The oocytes were continuously bathed (4 ml/min) with OR-2 buffer (in mM: 82.5 NaCl, 2.5 KCl, 1 Na2HPO4, 3 NaOH, 1 CaCl2, 1 MgCl2, 1 Na-pyruvate, 5 HEPES, pH 7.6), and the oocyte membrane potential was held at –60 mV. Buffer containing 400 µM PAH, with or without 0.5 mM probenecid, was applied to the oocytes in a pulsed manner via a solenoid-controlled valve. Recordings were sampled at 100 Hz with a 50-Hz filter frequency. After being sampled, current signals were processed with a Gaussian low band pass (2 Hz cut-off) filter. The current voltage (I-V) protocol was initiated from a –50-mV hold. Each holding potential was applied for 100 ms, and potentials were cycled from –150 to +50 mV in 20-mV increments, after which the clamping potential was returned to –50 mV. Steady-state currents were measured at 95 ms during each voltage pulse. I-V protocol recordings were sampled at 5 kHz with a 500-Hz filter frequency.

Cell Culture

MDCK type II cells (a low transepithelial resistance clone, originally derived from distal renal tubules of an adult female cocker spaniel) were provided by Dr. D. Balkovetz (University of Alabama at Birmingham) and were originally subcloned in the laboratory of Dr. K. Simons (EMBL, Heidelberg, Germany). They were negative for mycoplasma upon receipt. All MDCK lines were retested before publication and found to be negative for mycoplasma. Cells were maintained in Eagle's Modified Essential Medium (EMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum in a humidified incubator at 37°C with 5% CO2. Cultures were split 1:20 every 3–4 days.

Transfection

For mammalian cell transfection, the fragment containing the full-length hOAT1 cDNA was removed from the isolated library clone, pSPORT1/hOAT1 (6), with the restriction enzymes BamH 1 and Kpn 1. The fragment was gel-isolated and ligated into pcDNA3.1 (Invitrogen, Carlsbad, CA) cut with BamH 1 and Kpn 1, resulting in the plasmid pcDNA3.1/hOAT1. Activity of the new construct was confirmed by X. laevis oocyte expression assay before transfection. One day before transfection, 2 x 105 MDCK cells were plated into individual wells of a six-well culture plate (9.1 cm2). Cells were transfected with 10 µg pcDNA3.1/hOAT1 plasmid DNA for 3 h at 37°C using SuperFect Reagent (5 µl SuperFect/µg DNA; Qiagen, Chatsworth, CA). Transfected cells were washed with PBS, given fresh medium, and maintained at 37°C with 5% CO2. Two days after transfection, cells were lifted, diluted to ~1 cell/ml, dispensed (1 ml/well) into 24-well culture plates, and cultured in the presence of 1 mg/ml G418 (Invitrogen). Surviving cell clones were maintained with 200 µg/ml G418 and tested for organic anion transport activity.

Membrane Vesicles

Rat kidney BLM vesicles. Renal cortex was dissected from the kidneys of 15 rats, and BLM vesicles were isolated by differential and density (11% Percoll, Amersham Biosciences, Piscataway, NJ) gradient centrifugation as previously described (18). The final membrane pellet was suspended in KCl vesicle buffer [in mM: 100 mannitol, 100 KCl, 20 HEPES/Tris (hydroxymethyl) aminoethane (Tris), 1 MgSO4, at pH 7.5] and stored in liquid nitrogen until use. Upon thawing, vesicles were washed by centrifugation and resuspended in fresh buffer with additions as noted in the figure legends.

Plasma membranes from cultured MDCK cells. Cells were harvested by scraping and homogenized in buffer (in mM: 300 mannitol, 12 HEPES/Tris, and 0.1 phenylmethylsulphonyl fluoride) with 20 strokes using a pestle and glass homogenizer. The homogenate was centrifuged at 250 g for 15 min, and the pellet was discarded. The supernatant was centrifuged at 20,500 g for 20 min to collect the plasma membranes. The plasma membrane pellet was washed twice by resuspension and centrifugation at 20,500 g, and the final pellet was suspended in vesicle buffer and stored in liquid nitrogen. Marker enzyme analysis showed four- to sixfold enrichment of both brush border (alkaline phosphatase) and BLM (Na-K-ATPase) markers.

Vesicle Transport Measurement

Upon thawing, rat BLM and MDCK plasma membrane vesicles were centrifuged and resuspended in fresh vesicle buffer. Vesicles were allowed to equilibrate for 60 min at 22–24°C. Transport was measured using the rapid filtration method and Millipore HAWP (0.45 µm) membranes as previously described (12). Briefly, 10 µl of vesicles were incubated with 190 µl of vesicle buffer with additions as indicated in the figure legends. All vesicle experiments were conducted under short-circuiting conditions (100 mM KCl in = out plus 10 µM valinomycin). Uptake was terminated by addition of 1 ml of stop solution (in mM: 300 mannitol, 12 HEPES/Tris, and 0.1 HgCl2, at pH 7.5). PAH uptake values for all vesicle experiments (kinetic and static head experiments) are reported as probenecid-sensitive uptake. Radioactivity was determined using an LKB model 1216 liquid scintillation counter with external standard quench correction.

MDCK Cell Transport Assay

For transepithelial flux experiments, 1 x 106 cells were plated onto 24-mm (4.7 cm2) Transwell-Clear microporous polyester membranes (Costar, Bedford, MA). Cell monolayers were cultured for 3 days in EMEM supplemented with 10% fetal bovine serum without G418 (2 ml on each side of the monolayer) in a humidified incubator at 37°C with 5% CO2. The culture medium was changed daily. Before transport experiments, the culture medium was removed, the monolayers were washed twice with 2 ml of Hanks' balanced salt solution (Sigma) buffered with 10 mM HEPES (transport buffer), and 2 ml of transport buffer (pH 7.4) were added to both apical and basolateral compartments. Transport measurement was initiated by replacement of basolateral, apical, or both media with buffer containing 2 µM [3H]PAH (2 µCi/ml). After incubation at 37°C, the medium was removed from both sides of the monolayer and the cells were rapidly rinsed three times with ice-cold 0.1 M MgCl2. The cells were dissolved in 2 ml 1 N NaOH and neutralized with 2 ml 1 N HCl. Aliquots were removed for protein assay (2) using a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin used as a standard and for liquid scintillation counting using 15 ml of Ecolume (ICN Biomedical, Cleveland, OH). [3H]PAH uptake was normalized to cellular protein. For solid support experiments, 1 x 106 cells were plated into individual wells (3.5 cm2) of a 12-well tissue culture plate. Cells were handled exactly as described above for the experiments done on filter inserts, except that they were cultured for only 2 days after plating.

Estimation of Kinetic Parameters

The kinetic parameters for PAH uptake by hOAT1/MDCK cells were calculated by fitting the data to the following equation

where V is the uptake rate of PAH (pmol·min1·mg protein1), S is the PAH concentration in the medium (µM), Km is the apparent Michaelis Menten constant (µM), Vmax is the maximum uptake rate (pmol·min1·mg protein1), and Pdiff is the diffusion constant (µl·min1·mg protein1). Curve fitting was performed by an iterative nonlinear least-squares method using a MULTI program (31). The input data were weighed as the reciprocal of the observed values, and the Damping Gauss Newton Method was used as the fitting algorithm.

Statistics

Data are presented as means ± SE, and differences were considered to be significant when P < 0.05. Data between two experimental groups were compared using the unpaired Student's t-test.


    RESULTS
 TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
Electrophysiology

As shown in Fig. 1A, when rOat1-expressing X. laevis oocytes clamped at –60 mV were exposed to 400 µM PAH, net entry of positive charge was seen, ranging from 6 to 8 nA in multiple trials. This current was abolished by 0.5 mM probenecid. Reapplication of PAH after treatment with probenecid yielded a blunted response, probably because of lingering probenecid in the transporter binding sites. Control oocytes injected with water rather than rOat1 RNA showed no PAH or probenecid-induced current (data not shown). These results indicate that entry of PAH is accompanied by net entry of positive charge or loss of negative charge. Because rOat1 is known to mediate PAH/dicarboxylate exchange, the simplest interpretation consistent with this observation would be 1:1 exchange, i.e., of one monovalent organic anion for one divalent dicarboxylate anion. As shown in Fig. 1B, the current response of rOat1-expressing oocytes to 400 µM PAH was voltage sensitive. Inward positive current increased as the voltage was varied from –10 to –150 mV. Current flow reversed at –10 mV, and outward positive current was seen at voltages from –10 to +50 mV.



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Fig. 1. Electrophysiological recording of PAH-induced current (I) in a rat organic anion transporter (rOat)1-expressing oocyte. A: inward current (–60-mV clamp) observed in response to 400 µM PAH with and without 500 µM probenecid (Prob). B: rOat1 PAH (400 µM)-mediated current response to voltage steps. Responses are representative of results observed in at least 5 oocytes isolated from 3 different animals.

 

Rat BLM Vesicles

To directly test whether exchange was 1:1, kinetic (Hill analysis) and static head experiments were conducted in BLM vesicles isolated from rat renal cortex. As shown in Fig. 2, the basic experimental protocol was to measure the probenecid-sensitive uptake of [3H]PAH by vesicles preloaded with 1 mM glutarate and diluted 20-fold with transport buffer, i.e., an initial gradient of 1 mM glutarate inside vs. 50 µM outside. Under these conditions, a substantial glutarate-dependent overshoot was readily demonstrated.



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Fig. 2. Time course of probenecid-sensitive [3H]PAH uptake in control ({circ}) and glutarate-preloaded ({bullet}) rat renal cortex basolateral membrane vesicles (BLMV). BLMV were preloaded with 1 mM glutarate by incubation for 1 h. BLMV (10 µl) were then mixed with 190 µl vesicle buffer containing 50 µM [3H]PAH and 10 µM valinomycin in the presence and absence of 5 mM probenecid. Values are the means ± SE (n = 3 vesicle preparations analyzed in triplicate). *Significantly different from control, P < 0.05 by unpaired Student's t-test.

 

Kinetic Analysis

To determine the coupling ratio, the initial rate of PAH (50 µM) uptake was estimated at 15 s. Intravesicular glutarate was varied from 10 µM to 2 mM. As shown in Fig. 3A, probenecid-sensitive PAH uptake was a hyperbolic function of glutarate concentration and upon transformation according to the Hill equation (Fig. 3B), these data yielded a straight line with a slope (coupling coefficient) of 0.97 (Km = 94 µM; Vmax = 90 pmol·mg protein1·15 s1).



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Fig. 3. Kinetics of probenecid-sensitive glutarate exchange for [3H]PAH in rat renal cortex BLMV. BLMV were preloaded with glutarate (10 µM to 2 mM) as described in Fig. 2. BLMV were then diluted 1:20 with vesicle buffer containing 50 µM [3H]PAH and 10 µM valinomycin in the presence and absence of 5 mM probenecid. Uptake was measured at 15-s time points. A: probenecid-sensitive uptake of PAH as a function of the glutarate concentration. B: Hill transformation of these data. Hill coefficient = 0.97. Values are means ± SE (n = 3 vesicle preparations analyzed in triplicate). GA, glutamate.

 

Static Head

A second means of assessing the coupling between two transported species is to determine the gradients that balance one another and yield no net flux of substrate. This was done in short-circuited vesicles (100 mM KCl in = out, plus 10 µM valinomycin). For these experiments, the glutarate gradient was fixed at 10:1 in>out and, while maintaining a constant specific activity, in:out 3[H]PAH gradients of 1:1, 5:1, 10:1, 50:1, and 100:1 were tested. A coupling ratio of 1 PAH:1 glutarate would yield no net flux at equal gradients. As shown in Fig. 4, no net flux was observed when the gradients were 10:1 for both ions, indicating a 1:1 coupling ratio.



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Fig. 4. Static head analysis of PAH/glutarate exchange in rat renal cortex BLMV. BLMV were preloaded for 30 min in vesicle buffer (100 mM mannitol, 100 mM KCl, 20 mM Tris/HEPES at pH 7.5) containing 500 µM [3H]PAH and 500 µM glutarate. Vesicles were then diluted into vesicle buffer containing 10 µM valinomycin to achieve a 10-fold gradient for glutarate (50 µM outside; 500 µM inside), whereas the [3H]PAH gradient (with 500 µM inside) was varied from 1- to 100-fold (i.e., 500 µM to 5 µM outside). A constant specific activity was maintained throughout. The probenecid-sensitive [3H]PAH loss (efflux) or gain (influx) was then plotted as a function of [3H]PAH dilution. Values are means ± SE (n = 3 vesicle preparations analyzed in triplicate).

 

hOAT1-Transfected MDCK Cells

To determine whether membranes expressing a single cloned Oat would behave similarly to the native BLM in which multiple paralogs (e.g., Oat1 and Oat3) might be expressed, we generated an MDCK line stably transfected with hOAT1. As shown in Fig. 5, apical and basal uptake of PAH in hOAT1-expressing cells (hOAT1/MDCK) cultured on permeable filters increased as a function of time. Uptake of PAH by the parental, nontransfected MDCK cells was minimal and unaffected by other agents (not shown). When both faces of the epithelium were exposed simultaneously, uptake was larger than from either basal or apical sides alone and approximated their sum at all the time points. Clearly, hOAT1 expression in this clonal cell line is evident over the entire plasma membrane. Therefore, all subsequent characterization experiments were conducted using cells grown on solid support, i.e., assessing apically expressed hOAT1.



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Fig. 5. Accumulation of 2 µM [3H]PAH by stably transfected human (h)OAT1-expressing Madin-Darby canine kidney (MDCK) cells. Open bars indicate uptake from the basolateral face of the monolayer; gray bars show apical uptake; and black bars indicate uptake at both surfaces. Values are means ± SE (n = 3 experiments conducted in triplicate).

 

PAH uptake over 1 min by hOAT1/MDCK cells was saturable over the range of 0.5–200 µM (Fig. 6). The apparent Km in these hOAT1-expressing cells for PAH was 8.5 ± 1.3 µM with a Vmax 166 ± 21 pmol· min1·mg protein1 and a nonmediated component, Pdiff, of 0.47 ± 0.29 µl·min1·mg protein1 (Fig. 6). Uptake of 2 µM [3H]PAH by these cells was markedly inhibited by unlabeled PAH, glutarate, {alpha}-KG, probenecid, and uric acid (Fig. 7) in a manner very similar to our previous data on hOAT1 expressed in X. laevis oocytes (6). Fluorescein and the anionic herbicide, 2,4-D, also strongly inhibited PAH uptake. On the other hand, several larger organic anions including benzyl-penicillin, taurocholate, methotrexate, and leukotriene-C4 did not inhibit PAH uptake by the hOAT1/MDCK cells. Similarly, the p-glycoprotein substrate cyclosporin A and the organic cations tetraethylammonium and N1-methylnicotinamide did not significantly inhibit PAH uptake in the transfected cell line (Fig. 7). Substrate specificity of the hOAT1/MDCK cells was also evaluated directly by assessing uptake of radiolabeled glutarate, uric acid, and taurocholate. Figure 8A shows that glutarate (5 µM) is preferentially transported into hOAT1/MDCK cells compared with untransfected MDCK cell controls. In contrast, uptakes of uric acid (2 µM) and taurocholate (10 µM) were smaller than for glutarate and were not notably different between the transfected and untransfected lines (Fig. 8, B and C). Finally, both uptake (not shown) and efflux of PAH were trans-stimulated by 250 µM unlabeled PAH, glutarate, and {alpha}-KG, but not by TEA, at the same concentration, just as expected of an organic anion/dicarboxylate exchanger such as hOAT1 (Fig. 9).



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Fig. 6. Concentration dependence of [3H]PAH (0.5 to 200 µM) uptake in stably transfected hOAT1 MDCK cells. Samples were taken at 1 min to approximate the initial rate of uptake. Values are means ± SE (n = 3 experiments).

 


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Fig. 7. Inhibition of 2 µM [3H]PAH uptake (1 min) by stably transfected hOAT1-expressing MDCK cells. All compounds were tested at 200 µM except cyclosporin A (CSA; 10 µM) and leukotriene C4 (LTC4; 1 µM). Values are means ± SE (n = 2 experiments conducted in triplicate). *Significantly different from control, P < 0.05 by unpaired Student's t-test. {alpha}-KG, {alpha}-ketoglutarate; 2,4-D, 2-4-dichlorophenoxyacetic acid; TEA, tetraethylammonium; MTX, methotrexate; NMN, N1-methylnicotinamide.

 


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Fig. 8. Time course of accumulation (pmol/mg protein) of 5 µM [14C]glutarate (A), 2 µM [3H]-taurocholate (B), and 10 µM [14C]urate (C) in nontransfected (filled symbols) and stably transfected hOAT1 MDCK cells (open symbols). Values are means ± SE (n = 2 experiments conducted in triplicate). *Significantly different from nontransfected, P < 0.05 by unpaired Student's t-test.

 


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Fig. 9. Effect of external PAH, glutarate, {alpha}-KG, or TEA on [3H]PAH efflux from stably transfected hOAT1 MDCK cells. Cells were incubated with 2 µM [3H]PAH for 30 min at room temperature. The PAH-containing transport buffer was then removed, and the cells were rapidly rinsed twice with transport buffer. Cells were then incubated at 37°C with 2 ml transport buffer ±250 µM organic anion or cation. Total [3H]PAH cell content was calculated from the sum of total radioactivity recovered in the medium and that remaining in the cells at the conclusion of the 10-min efflux period. Values are means ± SE (n = 2 experiments conducted in triplicate). *Significantly different from control, P < 0.05 by unpaired Student's t-test.

 

Plasma Vesicles from hOAT1/MDCK Cells

Kinetic analysis. The preceding studies indicate clearly that hOAT1 expressed in this MDCK cell line has the properties expected of this transporter (Figs. 5, 6, 7, 8, 9). Thus plasma membrane vesicles prepared from these cells should show hOAT1 activity. When tested, vesicles preloaded with 1 mM glutarate and diluted 20-fold into medium containing 50 µM [3H]PAH exhibited a marked stimulation of PAH uptake with a transient overshoot of fivefold (data not shown), much the same as that depicted in Fig. 2 for native rat BLM vesicles. As shown in Fig. 10, the Hill plot for PAH/glutarate exchange conducted in the same manner as with rat renal cortex BLM vesicles yielded a slope of 0.97, indicating a coupling ratio of 1:1 for the hOAT1-expressing plasma membrane vesicles.



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Fig. 10. Hill-transformed kinetics of probenecid-sensitive [3H]PAH uptake in glutarate-preloaded plasma membrane vesicles prepared from stably transfected hOAT1/MDCK cells. Vesicles were preloaded with glutarate (10 µM to 2 mM) as described in Fig. 2. They were then diluted 20-fold in buffer containing 50 µM [3H]PAH and 10 µM valinomycin (±5 mM probenecid). Uptake was determined at 15-s time points. Hill = 0.97. Values are means ± SE (n = 3 vesicle preparations analyzed in triplicate).

 

Static head. hOAT1/MDCK cell plasma membrane vesicles were preloaded with glutarate and diluted 10-fold to create a 10:1 in>out gradient, and the net flux of [3H]PAH was determined at gradients from 1:1 to 100:1 in>out. Once again, as for the native membranes from rat kidney (Fig. 3), the cloned hOAT1-expressing membranes showed no net flux when both glutarate and PAH gradients were 10:1 (Fig. 11). Thus both kinetic and static head analysis of the expressed hOAT1 demonstrated a 1:1 coupling ratio.



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Fig. 11. Static head analysis of PAH/glutarate exchange in plasma membrane vesicles prepared from stably transfected hOAT1 MDCK cells. See Fig. 4 legend for details. Values are means ± SE (n = 3 vesicle preparations analyzed in triplicate).

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stoichiometry of Oat1

As shown in Fig. 1, accumulation of PAH by rOat1-expressing oocytes is accompanied by net entry of positive charge (or loss of negative charge). This result is comparable to that obtained by Burckhardt et al. (3) for winter flounder Oat1. Because basolateral organic anion transporters mediate exchange of singly charged anions such as PAH for doubly charged dicarboxylates (6, 22, 28), this result is most simply explained by a 1:1 coupling of organic anion (singly charged) and dicarboxylate (doubly charged) with the resulting net loss of one negative charge per transporter cycle. This conclusion was confirmed in native rat BLM vesicles and in plasma membranes isolated from MDCK cells expressing the human OAT1 ortholog, based on both Hill analysis (Figs. 3 and 10) and static head experiments (Figs. 4 and 11).

The similarity of findings for native rat BLMs and membranes expressing a single OAT paralog has particularly interesting implications. Clearly, based on Figs. 10 and 11, when hOAT1 was expressed in the absence of other Oats, it mediated 1:1 exchange of PAH for dicarboxylate. However, native rat BLMs express multiple Oat paralogs; the most prominent, in addition to Oat1, is Oat3 (9). Indeed, in the rat, Oat1 and Oat3 have very similar affinities for PAH [Km of 70 µM (28) and Km of 65 µM (10), respectively] and both should contribute to BLM vesicle PAH uptake. Certainly, recent findings in an Oat3 knockout mouse (27) indicate that this is the case for the mouse kidney, where Oat3 accounted for slightly more than half of the PAH uptake by renal slices. Thus our analysis of the rat membranes should have been influenced by both Oat paralogs, i.e., both would have contributed to the probenecid-sensitive uptake of PAH. In addition, we recently showed that Oat3 is, similar to Oat1, driven by organic anion/dicarboxylate exchange (24), meaning that a portion of the glutarate-driven, probenecid-sensitive PAH uptake observed in the rat BLM vesicles must have been driven by rOat3. Nevertheless, both Hill analysis and static head determination (Figs. 3 and 4) demonstrate that total probenecid-sensitive PAH uptake by the rat kidney BLM shows 1:1 coupling between PAH and glutarate. Thus, by implication, it appears that both Oat1 and Oat3 mediate PAH transport by 1:1 exchange for glutarate.

The electrophysiological results, the Hill analysis, and the static head experiments all provide a consistent answer to the question of coupling ratio. It is exchange of one PAH for one dicarboxylate2. Thus the overall exchange process is electrogenic with the net loss of one negative charge per transporter cycle. How then may the previous results showing a lack of sensitivity of PAH uptake to manipulation of membrane potential in vesicles (14, 20), intact tubules (30), and the cloned carrier (28) be explained? There are several possibilities, but at the moment, there is little evidence to support any of them. For example, a charged species such as chloride or proton might bind to and/or be transported during the carrier cycle. Indeed, our vesicle studies demonstrated a strong chloride dependence for PAH transport by rat basolateral vesicles (14). Also, an electrophysiological study in flounder Oat1-expressing oocytes revealed that a 10-fold reduction in bath chloride abolished PAH-mediated current and reduced PAH transport, strongly suggesting a role of chloride in Oat transport (4). However, the detailed studies of Schmitt and Burckhardt (20) in vesicles indicated that chloride was not cotransported with PAH and it was concluded that the effect of chloride was allosteric in nature. Another possible explanation may lie in the experimental approach of the earlier studies. In each case noted above (14, 20, 28, 30), the earlier studies examined the response of PAH transport following changes in membrane potential. Thus it is possible that what they show is that the rate-limiting step in the transport of PAH is not influenced by membrane potential. Thus, if the transport event is electrogenic, as shown here and in Burckhardt's studies of the flounder Oat1, it may be that the exchange of PAH for glutarate is not the rate-limiting step in the overall process. Other candidates for this role might be the on- or off-rate for substrate or counter-ion or perhaps an allosteric change in transporter structure allowing it to reorient in the membrane or to accept new substrate or counter-ion. Furthermore, it is also possible that differences in the degree of phosphorylation may alter transport properties. Resolution of these possibilities must await further study.

Transfected MDCK Cell Model

The hOAT1-expressing cell line used here shows saturable PAH uptake (Fig. 6), an inhibition pattern identical to that previously shown for this construct expressed in X. laevis oocytes (6) (Fig. 7), mediates glutarate uptake (Fig. 8), and demonstrates PAH/glutarate exchange (Fig. 9); all properties to be expected of an OAT1-expressing cell line. Furthermore, as shown in Fig. 5, hOAT1 is well expressed at the apical face of these cells. This is a significant technical advantage in that it allows uptake and efflux experiments to be conducted in cells grown on solid support. In addition, because the parent MDCK cell line shows virtually no probenecid-sensitive uptake of PAH (1) or glutarate (Fig. 8A), this model has very low background uptake of organic anions. Thus the hOAT1-expressing cell line used in these studies is well suited to mechanistic studies and for screening of toxicity related to OAT1-mediated transport (1).

Conclusions

The electrophysiological, kinetic, and static head experiments reported above all indicate that the stoichiometry of organic anion/dicarboxylate exchange by both rat and human OAT1 orthologs is 1:1 and that, contrary to earlier conclusions based on experimental manipulation of membrane potential, organic anion transport is an electrogenic process. In addition, these data highlight the utility of this MDCK cell line stably expressing hOAT1 in the absence of other OATs for studies characterizing the nature of hOAT1-mediated transport and/or its role in xenobiotic and drug toxicity.


    ACKNOWLEDGMENTS
 
Present address of Y.-H. Han: Dept. of Metabolism and Pharmacokinetics, Bristol-Meyers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. B. Pritchard, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (E-mail: pritcha3{at}niehs.nih.gov).

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


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