Transport of cimetidine by flounder and human renal organicanion transporter - 《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 13:50 |只看该作者 |倒序浏览 |打印

作者：Birgitta C.Burckhardt, StefanBrai, SönkeWallis, WolfgangKrick, Natascha A.Wolff,  GerhardBurckhardt作者单位：Zentrum Physiologie und Pathophysiologie, AbteilungVegetative Physiologie und Pathophysiologie, 37073 Göttingen,Germany

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   The H 2 -receptor antagonistcimetidine is efficiently excreted by the kidneys. In vivo studiesindicated an interaction of cimetidine not only with transporters forbasolateral uptake of organic cations but also with those involved inexcretion of organic anions. We therefore tested cimetidine as apossible substrate of the organic anion transporters cloned from winterflounder (fROAT) and from human kidney (hOAT1). Uptake of[ 3 H]cimetidine into fROAT-expressing Xenopuslaevis oocytes exceeded uptake into control oocytes. At 60-mVclamp potential, 1 mM cimetidine induced an inward current, which wassmaller than that elicited by 0.1 mM PAH. Cimetidine concentrationsexceeding 0.1 mM decreased PAH-induced inward currents, indicatinginteraction with the same transporter. At pH 6.6, no current wasseen with 0.1 mM cimetidine, whereas at pH 8.6 a current wasreadily detectable, suggesting preferential translocation of unchargedcimetidine by fROAT. Oocytes expressing hOAT1 also showed[ 3 H]cimetidine uptake. These data reveal cimetidine as asubstrate for fROAT/hOAT1 and suggest that organic anion transporterscontribute to cimetidine excretion in proximal tubules.   D* z) D  [, r2 d, {
      【关键词】 winter flounder renal organic anion transporter organic aniontransport organic cation transport kidney
   INTRODUCTION
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AS A SPECIFIC ANTAGONIST OF histamine H 2 receptors, cimetidine acts on gastricparietal cells to inhibit HCl secretion stimulated by histamine,pentagastrin, and acetylcholine. Between 50 and 80% of the doseadministered intravenously was recovered in urine as unchangedcimetidine, and elimination half-life was ~2 h in healthy volunteerswith normal kidney and hepatic function ( 24 ). The meansteady-state plasma concentration on a standard 1,000-mg daily dose was1 µg/ml (range 0.64-1.64 µg/ml). A renal clearance of 600 ml/min ( 24 ), exceeding the glomerular filtration rate byapproximately fivefold, indicated an efficient tubular secretion ofcimetidine. Therefore, cimetidine must interact with renal transporters, most probably with those located in the proximal tubules.
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Because at physiological pH part of the cimetidine is positivelycharged, the organic cation transport systems of the proximal tubulecell are expected to be involved in cimetidine secretion. The two-stepmodel of transcellular organic cation secretion consists ofelectrogenic uptake at the basolateral side, moving organic cationsdown their electrochemical gradient from blood into proximal tubularcells, and organic cation/proton exchange mediating uphill exit intothe lumen. The cloned organic cation transporters, OCT1, OCT2, andOCT3, are electrogenic and likely contribute to organic cation uptakeacross the basolateral membrane (for reviews, see Refs. 6 and 18 ). In accordance with their function, rat OCT1 and OCT2 werelocalized to the basolateral membrane of proximal tubule cells( 16, 25, 29 ). However, the precise location of OCT3 is notyet known. Tetraethylammonium uptake by rat ( 29 ) and human( 34 ) OCT1 was inhibited by unlabeled cimetidine in themedium with apparent K i values of 5.7 and 166 µM, respectively. Similarly, transport of 1-methyl-4-phenylpyridiniumby rabbit OCT1 was also inhibited by cimetidine ( 26 ).Translocation of radiolabeled cimetidine, however, was slow ornegligible in human and rat OCT1 ( 10, 34 ). Moreover,OCT1 / mice did not show reduced renal transport of cimetidinecompared with their wild-type littermates ( 14 ), suggestingthat OCT1 may not play a significant role in renal cimetidinesecretion. In contrast, rat OCT2 showed significant, saturabletransport of labeled cimetidine ( 10 ). The K m was 21 µM, a value close to the K i of 9.4 µM determined earlier for theinhibition of rat OCT2-mediated tetraethylammonium uptake by cimetidine( 29 ). In a chronic renal failure model, 5 6 nephrectomy, OCT2 expression and renal clearance of cimetidine werereduced, whereas the expression of OCT1 was unchanged in the remnantkidney ( 13 ). These data collectively suggested that OCT2plays a dominant role in cimetidine secretion in the rat.Tetraethylammonium transport by human OCT2 was inhibited by cimetidine,with a K i of 10.9 µM (at pH 7.0)( 1 ). The same K i (11 µM) wasfound for the inhibition of organic cation transport by isolated humanproximal tubules ( 22 ). Because the expression of OCT1 inthe human kidney is low ( 9, 33 ), OCT2 may well dominateOCT1 with respect to proximal tubular cimetidine transport. Aninteraction of cimetidine with mouse ( 31 ) and rat( 17, 32 ) OCT3 has also been demonstrated, and hence OCT3may add to overall cimetidine secretion, if it is indeed located in the basolateral membrane of proximal tubule cells.
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In the intact rat kidney, cimetidine inhibited the uptake not only of N 1 -methylnicotinamide, a prototypical substrateof organic cation transporters, but also of PAH, the classic substrateof the renal organic anion transporter ( 27, 28 ).Similarly, the uptake of radiolabeled cimetidine from capillaries intoproximal tubule cells was inhibited by tetraethylammonium andprobenecid, compounds known to interact with organic cation and organicanion transporters, respectively ( 27, 28 ). Analogous toorganic cations, secretion of organic anions is a two-step process. Fororganic anions, uptake across the basolateral membrane occurs againstan electrochemical potential difference, followed by an energeticallydownhill exit across the brush-border membrane ( 3 ). Of thecloned organic anion transporters, rat and human OAT1 and OAT3 havebeen localized to the basolateral membrane of proximal tubule cells,with OAT1 being expressed mainly in the S2 segment and OAT3 in allthree segments ( 4, 12, 15 ). Human ( 4 ) and rat( 7, 20 ) OAT3 transported radiolabeled cimetidine with K m values of 40 (rat OAT3) and 57 µM (humanOAT3). Unlabeled cimetidine inhibited the uptake of labeled organicanions by human ( 4, 15 ) and rat OAT3 ( 5, 7, 20 ), and a K i of 46.8 µM has beendetermined for rat OAT3 ( 21 ). Thus there is no doubt thatcimetidine is a substrate of human and rat OAT3. With regard toOAT1, conflicting results have been reported. Human OAT1 was inhibitedby cimetidine ( 15 ), whereas rat OAT1 was not significantlyaffected ( 21 ). So far, a translocation of labeledcimetidine by any OAT1 has not been shown, leaving open whether OAT1contributes to cimetidine secretion in proximal tubules.5 G$ P! f6 k0 s5 F1 Z7 S- w
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PAH and cimetidine are small, polar compounds, as indicated by theirstructure and their octanol-water partition coefficients (Fig. 1 ). At physiological pH, the COOH groupof PAH is negatively charged, whereas cimetidine is present as anuncharged as well as a positively charged molecule. Given ap K a of 6.9 ( 1 ), 76% of cimetidineis uncharged, and only 24% is positively charged at pH 7.4. Human OCT2has recently been shown to interact preferentially withpositively charged cimetidine ( 1 ). Secretion of theuncharged cimetidine should then be mediated by another transporter,possibly OAT1 and OAT3. Here, we report that flounder renal organicanion transporter, fROAT, and human OAT1 translocate cimetidine. These data indicate that both organic cation and anion transporters areinvolved in proximal tubular cimetidine secretion and allow for anefficient renal excretion of this drug.

Fig. 1. Chemical structures, p K a, and octanol-waterpartition coefficients (log P) of PAH and cimetidine as obtained fromRefs. 1, 8, and 24.. e# Z3 j0 W) [% A7 P5 H
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MATERIALS AND METHODS
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In vitro cRNA transcription. fROAT ( 30 ) and human OAT1 ( 23 ) cDNAs wereused as templates for cRNA synthesis. Plasmids were linearized with Not I, and in vitro cRNA transcription was performed using aT7 mMessage mMachine Kit (Ambion, Huntingdon, UK). The resulting cRNAswere resuspended in purified, RNAse-free water to a final concentration of 1-1.5 µg/µl.! E: @6 q% b7 D/ ^* K; j3 @
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Oocyte preparation and storage. Stage V and VI oocytes from Xenopus laevis (Nasco, FortAtkinson, WI) were treated with collagenase (type CLS II, Biochrom, Berlin, Germany) and maintained at 16-18°C in control solution (in mM): 90 NaCl, 3 KCl, 2 CaCl 2, 1 MgCl 2, and5 HEPES/Tris, pH 7.6. One day after removal, oocytes were injectedeither with 30 nl of cRNA (1 µg/µl) coding for fROAT or with 23 nlof cRNA (1.5 µg/µl) encoding for human OAT1 and maintained at16-18°C in control solution supplemented with 100 kU/IUpenicillin, 0.1 mg/l streptomycin, and 2.5 mM sodium pyruvate.After 4 days of incubation with daily medium changes, oocytes were usedfor electrophysiological as well as for tracer uptake studies. Oocytesinjected with water served as controls.
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Uptake and electrophysiological studies. Expression of fROAT and human OAT1 in oocytes was confirmed bycomparing the uptake of radiolabeled PAH between cRNA-injected oocytesand water-injected oocytes. Uptake of [ 3 H]PAH (5 µCi/ml; p -[glycyl-2- 3 H] aminohippuric acid)and [ 3 H]cimetidine (10 µCi/ml;[ N -methyl- 3 H]cimetidine) in oocytes wasassayed for 30 (fROAT) or 60 min (human OAT1) at room temperature incontrol solution additionally containing either 0.099 mM unlabeled and0.001 mM labeled PAH or 0.99 mM unlabeled and 0.01 mM labeledcimetidine. The incubation was stopped by aspiration of theincubation medium and several washes with ice-cold controlsolution, and the oocytes were assayed for radioactivity as describedby Wolff et al. ( 30 ).
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Electrophysiological studies were performed by the conventionaltwo-microelectrode, voltage-clamp method ( 2 ). Oocytes were superfused with control solution followed by the same mediumadditionally containing PAH or cimetidine. The membrane potential ofthe oocytes was clamped at 60 mV, and the current induced by 0.1 mMPAH was measured to demonstrate functional expression of the fROATprotein. Voltage pulses between 90 and  10 mV, in 10-mV increments,were applied for 5 s each, and steady-state currents were recorded to obtain current-voltage ( I - V ) relationships. Ingeneral, the I - V protocol was applied first undercontrol conditions and then 30 s after the superfusion solutionwas changed to the test solution. The difference between thesteady-state currents measured in the presence and absence ofsubstrates was considered as substrate-induced current.

Chemicals. All chemicals, including PAH and cimetidine were of analytic grade andpurchased from Merck (Darmstadt, Germany) or Sigma (Deisenhofen,Germany). [ 3 H]PAH and [ 3 H]cimetidine werefrom New England Nuclear (Cologne, Germany) and from Amersham PharmaciaBiotech (Buckinghamshire, UK), respectively. The concentrations ofuncharged and positively charged cimetidine were calculated using theHenderson-Hasselbalch equation and the p K a listed in Fig. 1.
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RESULTS

PAH- and cimetidine uptake into fROAT-expressing oocytes. To test whether the fROAT translocates cimetidine, X. laevis oocytes were injected with cRNA coding for fROAT. Four days later, these oocytes mediated uptake of 132.1 ± 15.7 pmol[ 3 H]PAH · 30 min 1 · oocyte 1 and 14.4 ± 4.23 pmol [ 3 H]cimetidine · 30 min 1 · oocyte 1.Water-injected control oocytes showed uptakes of 5.5 ± 0.8 pmol [ 3 H]PAH · 30 min 1 · oocyte 1 and2.7 ± 0.1 pmol[ 3 H]cimetidine · 30 min 1 · oocyte 1 (Fig. 2; representative experiment of a totalof 3 independent experiments, each with an average of 6-12oocytes/experimental condition). From these data, the fROAT-mediateduptake rates for [ 3 H]PAH and [ 3 H]cimetidineare 126.6 and 11.7 pmol · 30 min 1 · oocyte 1,respectively. Despite a 10 times lower substrate concentration, PAHuptake exceeded that of cimetidine by a factor of 10.8. Nevertheless, cimetidine was translocated in fROAT-expressing oocytes significantly more quickly than in water-injected control cells, indicating that thiscompound is a substrate for the organic anion transporter.

Fig. 2. [ 3 H]PAH- and[ 3 H]cimetidine-uptake in winter flounder organic aniontransporter (fROAT)-expressing oocytes. Oocytes were injected with cRNAcoding for fROAT, and 4 days later transport of 0.1 mM PAH (filledbars) or of 1 mM cimetidine (open bars) within an uptake period of 30 min was assayed. Water-injected oocytes were treated similarly. Arepresentative experiment of a total number of 3 independentexperiments, each with an average of 6-12 oocytes/experimentalcondition, is shown. * Significant difference betweenfROAT-expressing oocytes and water-injected controls, P, V2 }- E  l) e+ e& i+ V; e

Because the uptake of PAH through fROAT generated an inward current( 2 ), we tested next whether cimetidine is also able toinduce such a current. The two-electrode, voltage-clamp technique allows a direct comparison of PAH- and cimetidine-induced currents inthe same oocyte. As measured at a holding potential of 60 mV, inpaired experiments on 20 oocytes from 14 donors, application of 0.1 mMPAH and of 1 mM cimetidine resulted in inward currents of 22 ± 2 (data from Fig. 3 C ) and 4 ± 1 nA (data from Fig. 3 D ), respectively. Despitea 10 times lower concentration, the average PAH-induced currentexceeded that of cimetidine by a factor of 5.5. No such currents wereobserved in water-injected control oocytes (data not shown).

Fig. 3. PAH- and cimetidine-induced currents in oocytes expressing fROAT atpH 7.6. Steady-state currents were recorded in the absence( ) and presence ( ) of 0.1 mM PAH( A ) and in the absence ( ) and presence of 1 mM cimetidine (; B ). The holding potential( V c ) was 60 mV, and the hyper- anddepolarizing test pulses were applied for 5 s and ranged from 90to  10 mV in 10-mV increments. C : PAH-dependent currents( ), i.e., currents obtained in the presence of 0.1 mMPAH minus the currents obtained in the absence of 0.1 mM PAH. D : cimetidine-dependent currents ( ). Thedata were obtained in 20 oocytes from 14 donors in pairedexperiments.

The small inward currents observed on application of cimetidine couldbe due either to a low affinity of cimetidine for fROAT or to low ratesof translocation. To distinguish between these two possibilities, theconcentration whereby half-maximal saturation of the cimetidine-inducedcurrent was observed ( K 0.5 ) was determined. At 60 mV and pH 7.6, cimetidine-induced currents tended to saturate 250 µM. In three experiments, K 0.5 was 45 ± 9 µM, and the maximalcimetidine-induced current ( I max ) approached 2.92 ± 0.03 nA.
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Steady-state currents in fROAT-expressing oocytes were measured in theabsence and presence of PAH and are plotted as a function of membranepotential in Fig. 3 A. Respective currents in the absence andpresence of cimetidine are shown in Fig. 3 B. PAH and, to a much lesser extent, cimetidine increased the slope conductance, indicating electrogenic uptake. When the substrate-induced currents forPAH (Fig. 3 C ) and cimetidine (Fig. 3 D ) wereplotted as a function of membrane potential between 90 and  10 mV, alinear I - V relationship was obtained for PAH aswell as for cimetidine. Again, at all potentials, thecimetidine-induced currents were smaller than the PAH-induced currents.At pH 7.4, the reversal potential ( E rev ) for thePAH-induced current was 11.2 ± 2.4 mV (Fig. 3 C ) andthat for the cimetidine-induced current was 27.8 ± 4.6 mV (Fig. 3 D ).2 a" a& E& J: \$ D- |/ k
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Superposition of PAH- and cimetidine-induced currents. To test whether PAH and cimetidine utilize the same transporter, fROAT,we added increasing cimetidine concentrations in the presence of 0.1 mMPAH to fROAT-expressing oocytes. PAH plus cimetidine-induced currents( I ) were normalized to the PAH-induced current in the absence of cimetidine ( I 0 ) as shown in Fig. 4. The smallest cimetidine concentrationapplied (0.1 mM) increased the total current, whereas a furtherincrease in cimetidine concentration decreased it. At 5 mM cimetidine, found. Thus PAH- andcimetidine-induced currents are additive only at low, and nonadditiveat higher, cimetidine concentrations. The nonadditive behaviorindicates that PAH and cimetidine utilize the same transporter ratherthan two separate, electrogenic transporters in oocytes.# ^7 }* H7 I5 u& J7 G

Fig. 4. Influence of increasing cimetidine concentrations onPAH-induced inward current. The effect of cimetidine concentrationsfrom 0.1 to 5 mM was tested on the current evoked by 0.1 mM PAH at aclamp potential of 60 mV. Currents were normalized to currentsinduced by 0.1 mM PAH in the absence of cimetidine. Data were obtainedin 6 oocytes from 3 donors.) _& M. F$ I6 s+ p- R' r: l8 G8 n- }

Cimetidine-induced currents at pH 6.6 and 8.6. To examine the influence of uncharged cimetidine (CIM 0 ) onfROAT, cimetidine-induced currents at pH 6.6 and 8.6 were compared. AtpH 6.6 (Fig. 5 A, ), 0.1 mMtotal cimetidine did not induce a detectable current over the testedvoltage range, indicating that 0.033 mM CIM 0 wasinsufficient to induce measurable currents. At 1 mM, total cimetidine(0.333 mM CIM 0 ) evoked inward currents at membranepotentials more negative than 20 mV and outward currents at moredepolarized potentials. At pH 8.6 (Fig. 5 B ), comparablecurrents were induced by 0.1 (0.098 mM CIM 0 ) as well as by1 mM total cimetidine (0.98 mM CIM 0 ).9 N1 s0 `. J- i9 Q* z% q* A0 D5 I
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Fig. 5. Cimetidine-induced currents at pH 6.6 and 8.6. Currentsevoked by 0.1 ( ) or 1 mM cimetidine ( )were determined at pH 6.6 ( top ) and 8.6 ( bottom )as a function of clamp potential. Data were obtained in 6 oocytes from4 different donors.# u- Q8 ?' R% |- j! w
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[ 3 H]PAH and [ 3 H]cimetidine uptake byhuman OAT1-expressing oocytes. To explore whether cimetidine is also a substrate of human OAT1,oocytes were injected with cRNA coding for human OAT1 and assayed 4 days later. In two-electrode voltage-clamp studies, neither PAH norcimetidine induced an inward current. However, oocytes expressing humanOAT1 exhibited [ 3 H]PAH and [ 3 H]cimetidineuptake. Within 60 min, the [ 3 H]PAH- and the[ 3 H]cimetidine-associated uptakes were 43.6 ± 4.3 and 20.1 ± 4.3 pmol/oocyte, respectively, whereaswater-injected oocytes showed uptakes of 9.7 ± 1.1 and 6.8 ± 0.3 pmol/oocyte (Fig. 6;representative experiment of a total of 4, each with an average of6-15 oocytes/experimental condition). Thus human OAT1-mediateduptake rates were 33.9 and 13.3 pmol · 60 min 1 · oocyte 1 for[ 3 H]PAH and [ 3 H]cimetidine, respectively.The ratio of human OAT1-mediated PAH over cimetidine uptake was 2.5 inthese experiments.3 d8 \- \8 g+ t! O, U

Fig. 6. [ 3 H]PAH- and[ 3 H]cimetidine-uptake in human organic anion transporter1 (hOAT1)-expressing oocytes. Oocytes were injected with cRNA codingfor hOAT1, and 4 days later the uptake of 0.1 mM PAH (filled bars) orof 1 mM cimetidine (open bars) within 60 min was assayed.Water-injected oocytes were treated similarily. A representativeexperiment from 4 independent experiments with an average of 7-15oocytes/experimental condition is shown. * Significant differencebetween uptake of [ 3 H]PAH and[ 3 H]cimetidine compared with control, P& L# X: p/ E0 j% T% b4 E6 s& W6 v

DISCUSSION

In the rat kidney in situ, cimetidine uptake from the peritubularcapillaries into proximal tubule cells involved the organic cation andan organic anion transport system. Uptake of the organic cation N 1 -methylnicotinamide was inhibited bycimetidine with a K i of 0.16 mM and uptake ofthe organic anion PAH with a K i of 1.7 mM( 28 ). Thus the affinity of the organic cation transporterfor cimetidine was 10 times higher than that of the organic aniontransporter. The uptake of radiolabeled cimetidine from the capillarieswas inhibited by tetraethylammonium and probenecid, provingtranslocation by organic cation and anion transporters. As expectedfrom uptake through parallel systems, the inhibitions bytetraethylammonium and probenecid were additive. Interestingly,probenecid inhibited a larger portion of cimetidine uptake in situ thandid tetraethylammonium ( 28 ).

Also in the intact kidney, it was noted that organic cation andorganic anion transport systems "do not see the degree of substrateionization," because interaction with cimetidine with both systemswas rather unaffected, when capillary pH was changed between 6.0 and8.0 ( 27 ). Moreover, the uptake of labeled cimetidine wasthe same at pH 6.0, 7.4, and 8.0, suggesting that the transporters possibly interacted with both positively charged and uncharged cimetidine. These and related findings with dissociable primary, secondary, and tertiary amines led Ullrich and colleagues( 28 ) to postulate that substrates qualify for transport bythe presence of a hydrophobic core and by their ability to formhydrogen bonds.! ~: D* P3 M9 J0 o& r6 ^/ g
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Meanwhile, it is known that several transporters for organic ions existin the kidneys. Of the cloned organic cation transporters, OCT1 andOCT2 have been localized to the basolateral membrane of rat kidneyproximal tubule cells ( 16 ). Translocation of labeled cimetidine was slow or absent for human ( 34 ) and rat( 10 ) OCT1 but clearly demonstrable for expressed rat OCT2( 10 ). Thus OCT2 may contribute significantly to cimetidinesecretion, particularly in humans, where renal expression of OCT1 islow ( 6 ). Human OCT2 expressed in Chinese hamster ovary K1cells preferred the positively charged cimetidine over unchargedcimetidine: at higher pH, i.e., at a lesser abundance of chargedcimetidine (CIM ), human OCT2 showed a decreased affinityfor total (CIM 0 plus CIM ) cimetidine( 1 ).

Of the cloned organic anion transporters, OAT1 and OAT3 have beenlocalized to the basolateral membrane of proximal tubule cells in rat( 19 ) and human ( 4, 12 ) kidneys. Because both transport PAH, they may have contributed to the PAH transport asdetermined in the intact rat kidney. There is no doubt that rat andhuman OAT3 transport cimetidine ( 4, 7, 20 ). The interaction with the organic cation cimetidine has been taken as acharacteristic of OAT3. Mutational analysis of rat OAT3 revealed thatthe conserved cationic amino acids lysine 370 and arginine 454 areinvolved in the binding of PAH but not of cimetidine ( 7 ). Hydrophobic amino acid residues in the seventh transmembrane domain (tryptophan 334, phenylalanine 335, tyrosine 341, and other nearby residues) were found to be important for both PAH and cimetidine transport by allowing the formation of hydrogen bonds and hydrophobic interactions ( 8 ). Because these hydrophobic amino acidsare fully conserved in OAT1, this transporter could accept cimetidine as well.

X. laevis oocytes expressing fROAT translocated labeled PAH~12 times faster than did water-injected control oocytes. Transport of labeled cimetidine was also faster in fROAT-expressing oocytes, butthe transport ratio of fROAT-expressing to water-injected oocyteswas only four. In absolute terms, fROAT-mediated PAH uptake, i.e.,uptake in expressing minus uptake in water-injected oocytes was ~10times higher than uptake of cimetidine, although PAH concentration (0.1 mM) was one-tenth of the cimetidine concentration (1 mM). PAH andcimetidine induced inward currents. The current evoked by 0.1 mM PAHwas approximately five times higher than that induced by 1 mMcimetidine. If PAH and cimetidine were transported at equal rates,CIM 0 / -ketoglutarate 2 exchange wouldproduce an inward current twice as large asPAH / -ketoglutarate 2 exchange. However,because cimetidine uptake was only one-tenth of PAH uptake, thecimetidine-induced current should be one-fifth of the PAH-inducedcurrent. This was actually observed. Taken together, these data showthat fROAT-expressing oocytes translocate both PAH and cimetidine,albeit at considerably different rates.; Q  ~8 y+ s8 b$ H6 Y

To exclude that cimetidine uptake occurred through an endogenoustransporter upregulated by the expression of fROAT, we added PAH andcimetidine simultaneously to oocytes and measured the resulting inwardcurrent. If cimetidine was taken up by a separate endogenous,electrogenic transporter, PAH- and cimetidine-induced currents shouldbe additive at all cimetidine concentrations. If, however, PAH andcimetidine use the same carrier, increasing cimetidine concentrationsshould replace more and more PAH from fROAT. Because the maximumcurrent induced by saturating cimetidine concentrations is lower thanthat induced by PAH, replacement of PAH by cimetidine should reduce thetotal current. This was actually observed at cimetidine concentrationsexceeding 0.5 mM. Only at 0.1 mM cimetidine was a small increase intotal current observed, most likely due to translocation of cimetidineby transporter units not saturated with PAH. Taken together, ourexperiments provide direct evidence for transport of cimetidine by fROAT.; i3 M+ Q( A# J5 v+ G$ O* D$ v

The next question was whether CIM  , CIM 0, orboth forms of cimetidine interact with fROAT. To discriminate betweenthese possibilities, we changed bath pH from 7.6 to either 6.6 or 8.6. Given a p K a of 6.9, the relative abundance ofCIM 0 is 33.4, 83.4, and 98.0% at pH 6.6, 7.6, and 8.6, respectively. Conversely, CIM makes up 66.6, 16.6, and2.0% of total cimetidine at these pH values. If fROAT would preferCIM 0 over CIM  , currents should increase at aconstant cimetidine concentration when pH is raised. Unfortunately,fROAT itself is pH dependent, as seen with the anionic PAH as asubstrate: PAH-induced currents strongly decreased with increasing pH(data not shown), a behavior found earlier for PAH and adefovir uptakeby human OAT1 ( 11 ). Thus it is not possible toclearly discern the effects of pH on fROAT itself and on theavailability of the suited substrate (CIM 0 vs.CIM ). Taking a different approach, we added 0.1 or1 mM cimetidine at pH 6.6 and 8.6 to fROAT-expressing oocytes anddetermined the current. At pH 6.6, only 1 mM cimetidine evoked acurrent, whereas at pH 8.6 both concentrations induced similarcurrents. This result is best explained by assuming thatCIM 0 is preferred over CIM  . At pH 6.6 and 0.1 mM total cimetidine, CIM 0 concentration is 33 µM, whichwas obviously too low to elicit a measurable current. At 1 mM totalcimetidine, 333 µM CIM 0 amply sufficed to show inwardcurrents. At pH 8.6, 98 µM CIM 0 at 0.1 mM totalcimetidine concentration produced measurable currents as did 980 µMCIM 0 at 1 mM cimetidine. We emphasize that our data do notexclude that CIM may bind to, and be translocated by,fROAT with low affinity. However, the majority of cimetidine appears tobe transported as uncharged CIM 0. Thereby, fROAT does seethe degree of ionization. However, in the intact rat kidney, inhibitionof PAH uptake by cimetidine was pH independent ( 27 ). Apossible explanation is that PAH uptake in situ occurred through OAT1and OAT3 in parallel. If OAT1 and OAT3 show opposing preferences forCIM 0 and CIM  , the overall pH dependence ofcimetidine interaction with PAH transport should vanish.
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The I - V relationships of PAH- andcimetidine-induced currents reverse at inside negative membranepotentials. Although a negative E rev has alsobeen found in our earlier study ( 2 ), it remains to beclarified which ions contribute to PAH- and cimetidine-induced currents. Our first assumption was that the inward currents are due tothe electrogenic exchange of one PAH against one -ketoglutarate 2 molecule, causing the net efflux ofone negative charge. However, assuming that the PAH concentrationoutside the oocyte is higher than inside, and -ketoglutarateconcentration outside lower than inside, E rev should be far on the positive side. Replacement of PAH byCIM 0 or CIM would theoretically shift E rev to the left, i.e., to less positive values.Such a shift was indeed observed: E rev wasaround 10 mV for PAH-induced current and approximately 27 mV forcimetidine current (cf. Fig. 3 ). However, the problem remains that E rev was negative under both conditions. It ispossible that fROAT interacts, in addition to PAH (or cimetidine) and -ketoglutarate, with chloride or hydroxyl ions. Chloride removalinhibited PAH transport and abolished PAH-induced currents (data notshown), suggesting that chloride ions may modify or even betranslocated by fROAT. The large shift in E rev observed when bath pH was changed from 6.6 to 8.6 suggests thathydroxyl ions may be translocated by fROAT. However, more experimentsare needed to elucidate the ionic basis for the substrate-inducedcurrents generated in fROAT-expressing oocytes.1 B* S! z' N0 p! I7 G
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In the phylogenetic tree, fROAT is positioned between OAT1 and OAT3 andmay, therefore, combine transport properties of OAT1, i.e.,PAH/ -ketoglutarate exchange, and OAT3, i.e., interaction with bothorganic anions and cimetidine. Therefore, it was not clear a prioriwhether our findings can be extrapolated to mammals, particularly tohuman OAT1. We expressed human OAT1 in oocytes and found that ittransports both radiolabeled PAH and cimetidine. Relative to PAH,cimetidine transport was even higher in human OAT1 than in fROAT. Thustranslocation of cimetidine is no longer a specific feature of OAT3. Wetherefore propose that human OCT2, OAT1, and OAT3 contribute tocimetidine excretion. Most likely, OCT2 is responsible for the cationicform, whereas OAT1 takes care of the uncharged form of cimetidine,which actually predominates at pH 7.4. The preference of OAT3 is notyet known. Because probenecid inhibited a greater part of thecimetidine transport than did tetraethylammonium in the intact ratkidney ( 28 ), OAT1 and OAT3 may take the greater share. Insummary, the efficient renal excretion of cimetidine is due totransport through both organic cation and anion transporters in thebasolateral membrane of proximal tubule cells.
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ACKNOWLEDGEMENTS. R$ y  ~- _3 y$ M& x

The authors thank G. Dallmeyer and I. Markmann for technicalassistance and E. Thelen for the artwork.1 [0 \8 a5 z4 x
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