Stoichiometry of organic anion/dicarboxylate exchange in membrane vesicles from - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:45 |只看该作者 |倒序浏览 |打印

作者：Amy Aslamkhan, Yong-Hae Han, Ramsey Walden, Douglas H. Sweet,  John B. Pritchard作者单位：1 Laboratory of Pharmacology and Chemistry,National Institute of Environmental Health Sciences, National Institutes ofHealth, Research Triangle Park, North Carolina 27709; and Department of Pharmaceutical Sciences, MedicalUniversity of South Carolina, Charleston, South Carolina 29425 5 F' ]8 \8 }1 f9 d


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      【摘要】
   Although membrane vesicle studies have established the driving forces thatmediate renal organic anion secretion and the organic anion transporter Oat1has now been cloned in several species, its stoichiometry has remaineduncertain. In this study, we used electrophysiology, kinetic measurements, andstatic head experiments to determine the coupling ratio for Oat1-mediated organic anion/dicarboxylate exchange. Initial experiments demonstrated thatuptake of PAH by voltage-clamped Xenopus laevis oocytes expressingrOat1 led to net entry of positive charge, suggesting that coupling wasone-to-one. This conclusion was confirmed by kinetic analysis of PAH andglutarate fluxes in native basolateral membrane vesicles from the rat renalcortex, which showed a Hill coefficient of 1. Similarly, static headexperiments 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 humanexchanger hOAT1. The hOAT1-expressing cell line showed extensive PAHtransport, which was very similar in all respects to transport expressed byhOAT1 in Xenopus oocytes. Its K m for PAH was 8µM and glutarate effectively trans -stimulated PAH transport. Whenstoichiometry was assessed using plasma membranes isolated from thehOAT1-expressing cells, both kinetic and static head data indicated that hOAT1also demonstrated a 1:1 coupling between organic anion and dicarboxylate. 3 \* c' {0 |& e
      【关键词】 p aminohippurate hOAT organic anion transport anion exchange MadinDarby 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 forthe excretion of negatively charged chemicals and metabolites is the classicalorganic anion secretory system that transports PAH and other small anions ( cleared from therenal plasma in a single pass through the kidney. In the 1980s, membranevesicle studies led to the elucidation of the mechanisms and driving forcesthat enable this system to function so effectively( 14, 16, 23 ). The critical uphill stepin the secretory process was shown to be basolateral exchange of the anionicdrug or xenobiotic for intracellular -ketoglutarate ( -KG)( 15 ). Two organic aniontransporters, Oat1 and Oat3, have been localized to the basolateral membrane(BLM) ( 5, 7, 8, 25, 27 - 29 ).Of these, Oat1 was first to be cloned and demonstrated to be an organicanion/ -KG exchanger, initially in rats( 22, 28 ) and later in a number ofother species, including humans( 6, 8, 11, 19 ). Recently, we showed that rat Oat3 is also an organic anion/ -KG exchanger( 24 ).
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Surprisingly, given all of this attention, the stoichiometry of Oat1 hasremained uncertain. The original experiments conducted using membrane vesiclesisolated from rat kidney showed no change in PAH uptake when membranepotential was altered by changing potassium gradients in the presence of thepotassium ionophore valinomycin( 14 ). These data suggestedthat exchange was electroneutral, i.e., one divalent -KG molecule beingexchanged for two monovalent PAH molecules. Similar data were obtained inbovine vesicles ( 21 ), and acomparable conclusion was reached based on observations in the intact tubule( 30 ). Even in Xenopus laevis oocytes expressing the cloned rat transporter rOat1, no effect onPAH transport was observed when the oocytes were depolarized( 28 ). Because of theconsistency of these findings from different laboratories, the stoichiometrywas not determined directly. However, at least one study using rabbit renalBLM vesicles did show increased PAH uptake when membrane potential was insidenegative and decreased uptake when it was inside positive( 12 ). Furthermore, whenBurckhardt et al. ( 3 ) examined the electrophysiology of organic anion exchange in oocytes expressing winterflounder Oat1 under voltage-clamped conditions, entry of organic anions wasassociated with net entry of positive charge or exit of negative charge. Thisresult is most simply explained by a 1:1 exchange of organic anion fordicarboxylate. This issue, which has major implications in terms of transport mechanism at the molecular level, clearly requires resolution., Z( P/ z8 H: [8 n
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In the studies described below, we reexamined the stoichiometry ofOat1/OAT1 through 1 ) electrophysiological measurements in voltage-clamped rOat1-expressing Xenopus oocytes and 2 )kinetic measurements and static head experiments in native BLMs isolated fromrat kidney and in membranes isolated from Madin-Darby canine kidney (MDCK)cells stably expressing the human ortholog of the PAH/ -KG exchanger,hOAT1. In agreement with the electrophysiology data, the vesicle data showthat the coupling ratio of PAH to -KG is 1:1. Specifically, in bothmembrane preparations, the initial rate of PAH uptake was a hyperbolicfunction of the intravesicular concentration of counterion (Hill coefficient 1) and simultaneous 10-fold gradients for PAH and dicarboxylate balancedone another, yielding no net flux in static head experiments.
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METHODS AND MATERIALS

Chemicals; R( o) O2 S& _* P4 N
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[ 3 H]PAH (1.28 Ci/mmol) and [ 3 H]taurocholic acid (2.10Ci/mmol) were purchased from New England Nuclear (Boston, MA).[ 14 C]urate (55 mCi/mmol) and [ 14 C]glutarate (15.6mCi/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 commercialsources and were of the highest grade available.; r2 e2 c. \) y. S& z

Animals# a9 D: [; S0 D1 F

Rats. Kidneys were obtained from 300-g male Sprague-Dawley rats purchased from Taconic Farms (Germantown, NY) and maintained in the animalquarters at National Institute of Environmental Health Sciences (NIEHS) for 1wk or less. For tissue harvest, animals were euthanized with 100%CO 2 and the kidneys were immediately removed to ice-cold,oxygenated saline for preparation of isolated BLM.
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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 wereisolated by collagenase digestion as previously described( 6, 13, 26 ). All animal procedures were carried out in accord with protocols approved by the NIEHS Animal Careand Use Committee.

Electrophysiology% ^) U0 Y' k. l! m; C. \3 `

Three days after cRNA injection, oocyte membrane currents were measuredusing a conventional two-electrode (3 M KCl; resistance of 1 m )voltage-clamp technique (Geneclamp 500B, Axon Instruments, Foster City, CA).The oocytes were continuously bathed (4 ml/min) with OR-2 buffer (in mM: 82.5NaCl, 2.5 KCl, 1 Na 2 HPO 4, 3 NaOH, 1 CaCl 2, 1MgCl 2, 1 Na-pyruvate, 5 HEPES, pH 7.6), and the oocyte membranepotential was held at -60 mV. Buffer containing 400 µM PAH, with orwithout 0.5 mM probenecid, was applied to the oocytes in a pulsed manner via asolenoid-controlled valve. Recordings were sampled at 100 Hz with a 50-Hzfilter frequency. After being sampled, current signals were processed with aGaussian low band pass (2 Hz cut-off) filter. The current voltage (I-V)protocol was initiated from a -50-mV hold. Each holding potential wasapplied for 100 ms, and potentials were cycled from -150 to  50 mV in20-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 filterfrequency.
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Cell Culture% L! f. Q8 S7 O% t: \

MDCK type II cells (a low transepithelial resistance clone, originallyderived from distal renal tubules of an adult female cocker spaniel) wereprovided by Dr. D. Balkovetz (University of Alabama at Birmingham) and wereoriginally subcloned in the laboratory of Dr. K. Simons (EMBL, Heidelberg,Germany). They were negative for mycoplasma upon receipt. All MDCK lines wereretested before publication and found to be negative for mycoplasma. Cellswere maintained in Eagle's Modified Essential Medium (EMEM; Invitrogen,Carlsbad, CA) supplemented with 10% fetal bovine serum in a humidifiedincubator at 37°C with 5% CO 2. Cultures were split 1:20 every3-4 days.
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Transfection

For mammalian cell transfection, the fragment containing the full-lengthhOAT1 cDNA was removed from the isolated library clone, pSPORT1/hOAT1( 6 ), with the restrictionenzymes 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 inthe plasmid pcDNA3.1/hOAT1. Activity of the new construct was confirmed by X. laevis oocyte expression assay before transfection. One day beforetransfection, 2 x 10 5 MDCK cells were plated into individualwells of a six-well culture plate (9.1 cm 2 ). Cells were transfectedwith 10 µg pcDNA3.1/hOAT1 plasmid DNA for 3 h at 37°C using SuperFect Reagent (5 µl SuperFect/µg DNA; Qiagen, Chatsworth, CA). Transfectedcells were washed with PBS, given fresh medium, and maintained at 37°Cwith 5% CO 2. 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 cellclones were maintained with 200 µg/ml G418 and tested for organic aniontransport activity.
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Membrane Vesicles" o% i( g3 z2 o/ ^- e# u* t

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 aspreviously described ( 18 ). The final membrane pellet was suspended in KCl vesicle buffer [in mM: 100mannitol, 100 KCl, 20 HEPES/Tris (hydroxymethyl) aminoethane (Tris), 1MgSO 4, at pH 7.5] and stored in liquid nitrogen until use. Uponthawing, vesicles were washed by centrifugation and resuspended in freshbuffer with additions as noted in the figure legends.

Plasma membranes from cultured MDCK cells. Cells were harvested byscraping and homogenized in buffer (in mM: 300 mannitol, 12 HEPES/Tris, and0.1 phenylmethylsulphonyl fluoride) with 20 strokes using a pestle and glasshomogenizer. The homogenate was centrifuged at 250 g for 15 min, andthe pellet was discarded. The supernatant was centrifuged at 20,500 g for 20 min to collect the plasma membranes. The plasma membrane pellet waswashed 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.9 l( d; ]4 j3 D5 u1 I

Vesicle Transport Measurement

Upon thawing, rat BLM and MDCK plasma membrane vesicles were centrifugedand resuspended in fresh vesicle buffer. Vesicles were allowed to equilibratefor 60 min at 22-24°C. Transport was measured using the rapidfiltration method and Millipore HAWP (0.45 µm) membranes as previouslydescribed ( 12 ). Briefly, 10µl of vesicles were incubated with 190 µl of vesicle buffer withadditions as indicated in the figure legends. All vesicle experiments wereconducted under short-circuiting conditions (100 mM KCl in = out plus 10 µMvalinomycin). Uptake was terminated by addition of 1 ml of stop solution (inmM: 300 mannitol, 12 HEPES/Tris, and 0.1 HgCl 2, at pH 7.5). PAHuptake values for all vesicle experiments (kinetic and static headexperiments) are reported as probenecid-sensitive uptake. Radioactivity wasdetermined using an LKB model 1216 liquid scintillation counter with external standard quench correction.
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MDCK Cell Transport Assay% R( ]& N8 I7 p3 \3 Q5 ~8 E

For transepithelial flux experiments, 1 x 10 6 cells wereplated onto 24-mm (4.7 cm 2 ) Transwell-Clear microporous polyester membranes (Costar, Bedford, MA). Cell monolayers were cultured for 3 days inEMEM supplemented with 10% fetal bovine serum without G418 (2 ml on each sideof the monolayer) in a humidified incubator at 37°C with 5%CO 2. The culture medium was changed daily. Before transportexperiments, the culture medium was removed, the monolayers were washed twicewith 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 bothapical and basolateral compartments. Transport measurement was initiated byreplacement of basolateral, apical, or both media with buffer containing 2µM [ 3 H]PAH (2 µCi/ml). After incubation at 37°C, themedium was removed from both sides of the monolayer and the cells were rapidlyrinsed three times with ice-cold 0.1 M MgCl 2. The cells weredissolved 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) withbovine serum albumin used as a standard and for liquid scintillation counting using 15 ml of Ecolume (ICN Biomedical, Cleveland, OH). [ 3 H]PAH uptake was normalized to cellular protein. For solid support experiments, 1 x 10 6 cells were plated into individual wells (3.5cm 2 ) of a 12-well tissue culture plate. Cells were handled exactlyas described above for the experiments done on filter inserts, except thatthey were cultured for only 2 days after plating.+ z! l2 |! V' b! ~, p( F4 a; z

Estimation of Kinetic Parameters

The kinetic parameters for PAH uptake by hOAT1/MDCK cells were calculatedby fitting the data to the following equation* @5 b" k9 t0 P; W7 e
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where V is the uptake rate of PAH(pmol·min - 1 ·mg protein - 1 ), S is the PAH concentration in themedium (µM), K m is the apparent Michaelis Mentenconstant (µM), V max is the maximum uptake rate(pmol·min - 1 ·mg protein - 1 ), and P diff is thediffusion constant (µl·min - 1 ·mg protein - 1 ). Curve fitting was performed by aniterative nonlinear least-squares method using a MULTI program( 31 ). The input data wereweighed as the reciprocal of the observed values, and the Damping Gauss NewtonMethod was used as the fitting algorithm.

Statistics

Data are presented as means ± SE, and differences were considered tobe significant when P two experimental groupswere compared using the unpaired Student's t -test.

RESULTS

Electrophysiology0 H4 S" F" ?1 \9 j4 @' p
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As shown in Fig.1 A, when rOat1-expressing X. laevis oocytes clamped at -60 mV were exposed to 400 µM PAH, net entry of positivecharge was seen, ranging from 6 to 8 nA in multiple trials. This current wasabolished by 0.5 mM probenecid. Reapplication of PAH after treatment withprobenecid yielded a blunted response, probably because of lingeringprobenecid in the transporter binding sites. Control oocytes injected with water rather than rOat1 RNA showed no PAH or probenecid-induced current (datanot shown). These results indicate that entry of PAH is accompanied by netentry of positive charge or loss of negative charge. Because rOat1 is known tomediate PAH/dicarboxylate exchange, the simplest interpretation consistentwith this observation would be 1:1 exchange, i.e., of one monovalent organic anion for one divalent dicarboxylate anion. As shown in Fig. 1 B, the currentresponse 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.

Fig. 1. Electrophysiological recording of PAH-induced current ( I ) in a ratorganic anion transporter (rOat)1-expressing oocyte. A : inwardcurrent (-60-mV clamp) observed in response to 400 µM PAH with andwithout 500 µM probenecid (Prob). B : rOat1 PAH (400µM)-mediated current response to voltage steps. Responses arerepresentative of results observed in at least 5 oocytes isolated from 3different animals.& Y" N) d' j0 L) p5 \

Rat BLM Vesicles

To directly test whether exchange was 1:1, kinetic (Hill analysis) andstatic head experiments were conducted in BLM vesicles isolated from rat renalcortex. As shown in Fig. 2, thebasic experimental protocol was to measure the probenecid-sensitive uptake of[ 3 H]PAH by vesicles preloaded with 1 mM glutarate and diluted20-fold with transport buffer, i.e., an initial gradient of 1 mM glutarateinside vs. 50 µM outside. Under these conditions, a substantialglutarate-dependent overshoot was readily demonstrated.; B: Z! R* H8 D

Fig. 2. Time course of probenecid-sensitive [ 3 H]PAH uptake in control( ) and glutarate-preloaded ( ) rat renal cortex basolateral membranevesicles (BLMV). BLMV were preloaded with 1 mM glutarate by incubation for 1h. BLMV (10 µl) were then mixed with 190 µl vesicle buffer containing 50µM [ 3 H]PAH and 10 µM valinomycin in the presence and absenceof 5 mM probenecid. Values are the means ± SE ( n = 3 vesiclepreparations analyzed in triplicate). *Significantly different from control, P t -test.2 b" g! d* K& n# G  F

Kinetic Analysis

To determine the coupling ratio, the initial rate of PAH (50 µM) uptakewas estimated at 15 s. Intravesicular glutarate was varied from 10 µM to 2mM. As shown in Fig.3 A, probenecid-sensitive PAH uptake was a hyperbolicfunction of glutarate concentration and upon transformation according to theHill equation ( Fig.3 B ), these data yielded a straight line with a slope(coupling coefficient) of 0.97 ( K m = 94 µM; V max = 90 pmol·mgprotein - 1 ·15s - 1 ).

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

Static Head
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A second means of assessing the coupling between two transported species isto determine the gradients that balance one another and yield no net flux ofsubstrate. This was done in short-circuited vesicles (100 mM KCl in = out,plus 10 µM valinomycin). For these experiments, the glutarate gradient wasfixed at out and, while maintaining a constant specific activity, in

ut 3 PAH gradients of 1:1, 5:1, 10:1, 50:1, and 100:1 weretested. A coupling ratio of 1 PAH:1 glutarate would yield no net flux at equalgradients. 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 [ 3 H]PAH and 500µM glutarate. Vesicles were then diluted into vesicle buffer containing 10µM valinomycin to achieve a 10-fold gradient for glutarate (50 µMoutside; 500 µM inside), whereas the [ 3 H]PAH gradient (with 500µM inside) was varied from 1- to 100-fold (i.e., 500 µM to 5 µMoutside). A constant specific activity was maintained throughout. Theprobenecid-sensitive [ 3 H]PAH loss (efflux) or gain (influx) wasthen plotted as a function of [ 3 H]PAH dilution. Values are means± SE ( n = 3 vesicle preparations analyzed in triplicate).- C( e, m) _7 u; p) t( y1 N4 n
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hOAT1-Transfected MDCK Cells' Y6 t% s8 o7 i8 s) K& g
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To determine whether membranes expressing a single cloned Oat would behavesimilarly 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 andbasal uptake of PAH in hOAT1-expressing cells (hOAT1/MDCK) cultured onpermeable filters increased as a function of time. Uptake of PAH by theparental, 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 andapproximated their sum at all the time points. Clearly, hOAT1 expression inthis clonal cell line is evident over the entire plasma membrane. Therefore,all subsequent characterization experiments were conducted using cells grownon solid support, i.e., assessing apically expressed hOAT1.: p% g5 g& Z$ N# }5 h7 D7 X8 x" h" e

Fig. 5. Accumulation of 2 µM [ 3 H]PAH by stably transfected human(h)OAT1-expressing Madin-Darby canine kidney (MDCK) cells. Open bars indicateuptake from the basolateral face of the monolayer; gray bars show apicaluptake; and black bars indicate uptake at both surfaces. Values are means± SE ( n = 3 experiments conducted in triplicate).
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PAH uptake over 1 min by hOAT1/MDCK cells was saturable over the range of0.5-200 µM ( Fig. 6 ).The apparent K m in these hOAT1-expressing cells for PAHwas 8.5 ± 1.3 µM with a V max 166 ± 21pmol· min - 1 ·mg protein - 1 and a nonmediated component, P diff, of 0.47 ± 0.29µl·min - 1 ·mgprotein - 1 ( Fig.6 ). Uptake of 2 µM [ 3 H]PAH by these cells was markedly inhibited by unlabeled PAH, glutarate, -KG, probenecid, anduric acid ( Fig. 7 ) in a mannervery similar to our previous data on hOAT1 expressed in X. laevis oocytes ( 6 ). Fluorescein andthe anionic herbicide, 2,4-D, also strongly inhibited PAH uptake. On the otherhand, several larger organic anions including benzyl-penicillin, taurocholate,methotrexate, and leukotriene-C 4 did not inhibit PAH uptake by thehOAT1/MDCK cells. Similarly, the p-glycoprotein substrate cyclosporin A andthe organic cations tetraethylammonium and N 1 -methylnicotinamide did not significantly inhibit PAHuptake in the transfected cell line ( Fig.7 ). Substrate specificity of the hOAT1/MDCK cells was alsoevaluated directly by assessing uptake of radiolabeled glutarate, uric acid,and taurocholate. Figure8 A shows that glutarate (5 µM) is preferentiallytransported into hOAT1/MDCK cells compared with untransfected MDCK cellcontrols. In contrast, uptakes of uric acid (2 µM) and taurocholate (10µM) were smaller than for glutarate and were not notably different betweenthe 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 -KG, but not by TEA, at the same concentration, just as expected of anorganic anion/dicarboxylate exchanger such as hOAT1( Fig. 9 ).
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Fig. 6. Concentration dependence of [ 3 H]PAH (0.5 to 200 µM) uptake instably transfected hOAT1 MDCK cells. Samples were taken at 1 min toapproximate the initial rate of uptake. Values are means ± SE( n = 3 experiments)." }+ Y' N& I/ ?3 D: L
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Fig. 7. Inhibition of 2 µM [ 3 H]PAH uptake (1 min) by stablytransfected hOAT1-expressing MDCK cells. All compounds were tested at 200µM except cyclosporin A (CSA; 10 µM) and leukotriene C 4 (LTC4; 1 µM). Values are means ± SE ( n = 2 experimentsconducted in triplicate). *Significantly different from control, P t -test. -KG, -ketoglutarate; 2,4-D, 2-4-dichlorophenoxyacetic acid; TEA,tetraethylammonium; MTX, methotrexate; NMN, N 1 -methylnicotinamide.. k' k8 a" r) J4 [% b

Fig. 8. Time course of accumulation (pmol/mg protein) of 5 µM[ 14 C]glutarate ( A ), 2 µM [ 3 H]-taurocholate( B ), and 10 µM [ 14 C]urate ( C ) innontransfected (filled symbols) and stably transfected hOAT1 MDCK cells (opensymbols). Values are means ± SE ( n = 2 experiments conductedin triplicate). *Significantly different from nontransfected, P t -test.! t; ~# L  [3 l5 [1 ?8 X# T* p' V

Fig. 9. Effect of external PAH, glutarate, -KG, or TEA on [ 3 H]PAHefflux from stably transfected hOAT1 MDCK cells. Cells were incubated with 2µM [ 3 H]PAH for 30 min at room temperature. The PAH-containingtransport buffer was then removed, and the cells were rapidly rinsed twicewith transport buffer. Cells were then incubated at 37°C with 2 mltransport buffer ±250 µM organic anion or cation. Total[ 3 H]PAH cell content was calculated from the sum of totalradioactivity recovered in the medium and that remaining in the cells at theconclusion of the 10-min efflux period. Values are means ± SE( n = 2 experiments conducted in triplicate). *Significantly differentfrom control, P t -test.

Plasma Vesicles from hOAT1/MDCK Cells- Q  I) Q7 o* R( n
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Kinetic analysis. The preceding studies indicate clearly that hOAT1 expressed in this MDCK cell line has the properties expected of thistransporter (Figs. 5, 6, 7, 8, 9 ). Thus plasma membranevesicles prepared from these cells should show hOAT1 activity. When tested,vesicles preloaded with 1 mM glutarate and diluted 20-fold into mediumcontaining 50 µM [ 3 H]PAH exhibited a marked stimulation of PAHuptake with a transient overshoot of fivefold (data not shown), much the sameas that depicted in Fig. 2 fornative rat BLM vesicles. As shown in Fig.10, the Hill plot for PAH/glutarate exchange conducted in the samemanner 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 membranevesicles.
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Fig. 10. Hill-transformed kinetics of probenecid-sensitive [ 3 H]PAH uptakein glutarate-preloaded plasma membrane vesicles prepared from stablytransfected hOAT1/MDCK cells. Vesicles were preloaded with glutarate (10 µMto 2 mM) as described in Fig.2. They were then diluted 20-fold in buffer containing 50 µM[ 3 H]PAH and 10 µM valinomycin (±5 mM probenecid). Uptakewas determined at 15-s time points. Hill = 0.97. Values are means ± SE( n = 3 vesicle preparations analyzed in triplicate).
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Static head. hOAT1/MDCK cell plasma membrane vesicles were preloaded with glutarate and diluted 10-fold to create a 10:1 outgradient, and the net flux of [ 3 H]PAH was determined out. Once again, as for the native membranes from ratkidney ( Fig. 3 ), the cloned hOAT1-expressing membranes showed no net flux when both glutarate and PAHgradients were 10:1 ( Fig. 11 ).Thus both kinetic and static head analysis of the expressed hOAT1 demonstrateda 1:1 coupling ratio.

Fig. 11. Static head analysis of PAH/glutarate exchange in plasma membrane vesiclesprepared from stably transfected hOAT1 MDCK cells. See Fig. 4 legend for details.Values are means ± SE ( n = 3 vesicle preparations analyzed intriplicate).5 g* K; M$ b. A# M3 o+ L: r; E  q

DISCUSSION9 H: n: u0 d9 _: @$ ~: I7 w; y! k: @

Stoichiometry of Oat1, B7 z' a' [; v1 X! G) X

As shown in Fig. 1,accumulation of PAH by rOat1-expressing oocytes is accompanied by net entry ofpositive charge (or loss of negative charge). This result is comparable tothat obtained by Burckhardt et al.( 3 ) for winter flounder Oat1. Because basolateral organic anion transporters mediate exchange of singlycharged anions such as PAH for doubly charged dicarboxylates ( 6, 22, 28 ), this result is mostsimply explained by a 1:1 coupling of organic anion (singly charged) anddicarboxylate (doubly charged) with the resulting net loss of one negative charge per transporter cycle. This conclusion was confirmed in native rat BLMvesicles and in plasma membranes isolated from MDCK cells expressing the humanOAT1 ortholog, based on both Hill analysis (Figs. 3 and 10 ) and static headexperiments (Figs. 4 and 11 ).% ~6 W  j/ A# J1 i% n
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The similarity of findings for native rat BLMs and membranes expressing asingle OAT paralog has particularly interesting implications. Clearly, basedon Figs. 10 and 11, when hOAT1 was expressedin the absence of other Oats, it mediated 1:1 exchange of PAH fordicarboxylate. However, native rat BLMs express multiple Oat paralogs; themost prominent, in addition to Oat1, is Oat3( 9 ). Indeed, in the rat, Oat1and Oat3 have very similar affinities for PAH [ K m of 70µM ( 28 ) and K m of 65 µM( 10 ), respectively] and bothshould 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 forslightly more than half of the PAH uptake by renal slices. Thus our analysisof the rat membranes should have been influenced by both Oat paralogs, i.e.,both would have contributed to the probenecid-sensitive uptake of PAH. Inaddition, we recently showed that Oat3 is, similar to Oat1, driven by organicanion/dicarboxylate exchange ( 24 ), meaning that a portionof the glutarate-driven, probenecid-sensitive PAH uptake observed in the ratBLM vesicles must have been driven by rOat3. Nevertheless, both Hill analysisand static head determination (Figs. 3 and 4 ) demonstrate that total probenecid-sensitive PAH uptake by the rat kidney BLM shows 1:1 couplingbetween PAH and glutarate. Thus, by implication, it appears that both Oat1 andOat3 mediate PAH transport by 1:1 exchange for glutarate.* v) Q+ A! u! y8 i1 G7 p# y
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The electrophysiological results, the Hill analysis, and the static headexperiments all provide a consistent answer to the question of coupling ratio.It is exchange of one PAH - for onedicarboxylate - 2. Thus the overall exchange process is electrogenic with the net loss of one negative charge per transportercycle. How then may the previous results showing a lack of sensitivity of PAHuptake to manipulation of membrane potential in vesicles( 14, 20 ), intact tubules( 30 ), and the cloned carrier( 28 ) be explained? There areseveral possibilities, but at the moment, there is little evidence to supportany of them. For example, a charged species such as chloride or proton mightbind to and/or be transported during the carrier cycle. Indeed, our vesiclestudies demonstrated a strong chloride dependence for PAH transport by ratbasolateral vesicles ( 14 ). Also, an electrophysiological study in flounder Oat1-expressing oocytesrevealed that a 10-fold reduction in bath chloride abolished PAH-mediatedcurrent and reduced PAH transport, strongly suggesting a role of chloride inOat transport ( 4 ). However, thedetailed studies of Schmitt and Burckhardt( 20 ) in vesicles indicatedthat chloride was not cotransported with PAH and it was concluded that theeffect of chloride was allosteric in nature. Another possible explanation maylie in the experimental approach of the earlier studies. In each case notedabove ( 14, 20, 28, 30 ), the earlier studiesexamined the response of PAH transport following changes in membranepotential. 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, ifthe transport event is electrogenic, as shown here and in Burckhardt's studiesof the flounder Oat1, it may be that the exchange of PAH for glutarate is notthe 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 anallosteric change in transporter structure allowing it to reorient in themembrane or to accept new substrate or counter-ion. Furthermore, it is alsopossible that differences in the degree of phosphorylation may alter transportproperties. Resolution of these possibilities must await further study.4 k- w" u- @  E8 C  |% Y  k
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Transfected MDCK Cell Model
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The hOAT1-expressing cell line used here shows saturable PAH uptake( Fig. 6 ), an inhibition patternidentical to that previously shown for this construct expressed in X.laevis oocytes ( 6 ) ( Fig. 7 ), mediates glutarateuptake ( Fig. 8 ), anddemonstrates 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. Thisis a significant technical advantage in that it allows uptake and effluxexperiments to be conducted in cells grown on solid support. In addition,because the parent MDCK cell line shows virtually no probenecid-sensitiveuptake of PAH ( 1 ) or glutarate( Fig. 8 A ), this modelhas very low background uptake of organic anions. Thus the hOAT1-expressingcell line used in these studies is well suited to mechanistic studies and forscreening of toxicity related to OAT1-mediated transport ( 1 ).! S9 J1 G* V  I' I: \6 j

Conclusions* d# |% ]2 p. `# }

The electrophysiological, kinetic, and static head experiments reportedabove all indicate that the stoichiometry of organic anion/dicarboxylateexchange by both rat and human OAT1 orthologs is 1:1 and that, contrary toearlier conclusions based on experimental manipulation of membrane potential,organic anion transport is an electrogenic process. In addition, these datahighlight the utility of this MDCK cell line stably expressing hOAT1 in theabsence of other OATs for studies characterizing the nature of hOAT1-mediatedtransport and/or its role in xenobiotic and drug toxicity.% c. b7 E# w  M6 P, J7 c* E- o
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ACKNOWLEDGMENTS
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Present address of Y.-H. Han: Dept. of Metabolism and Pharmacokinetics, Bristol-Meyers Squibb Pharmaceutical Research Institute, Princeton, NJ08543-4000.
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