Regulation of human organic cation transporter hOCT2 by PKA,PI3K, and calmodulin - 《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 13:32 |只看该作者 |倒序浏览 |打印

作者：IbrahimÇetinkaya, GiulianoCiarimboli, GülayYalçinkaya, ThomasMehrens, AnaVelic, Jochen R.Hirsch, ValentinGorboulev, HermannKoepsell,  EberhardSchlatter作者单位：1 Medizinische Klinik und Poliklinik D,Experimentelle Nephrologie, and Klinik und Poliklinikfür Kinderheilkunde, Universitätsklinikum Münster,D-48149 Münster; and Institut fürAnatomie und Zellbiologie, Universität Würzburg,D-97070 Würzburg, Germany



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      【摘要】! W; c9 d' D+ C" X2 |
   Properties and regulation ofthe human organic cation (OC) transporter type 2 (hOCT2) expressed inHEK-293 cells were extensively characterized using the fluorescent OC4-[4-(dimethylamino)styryl]- N -methylpyridinium (ASP ). ASP uptake was electrogenic andinhibited by TPA (EC 50 = 2.7 µM),tetraethylammonium (EC 50 = 35 µM), cimetidine (EC 50 = 36 µM), or quinine (EC 50 = 6.7 µM). Stimulation with carbachol or ATP decreased initial uptakeby 44 ± 3 ( n = 14) and 34 ± 4% ( n = 21), respectively, independently of PKC butdependent on phosphatidylinositol 3-kinase (PI3K). PKA stimulationdecreased uptake by 18 ± 4% ( n = 40). Inhibitionof calmodulin (CaM), Ca 2  /CaM-dependent kinase II, ormyosin light chain kinase decreased uptake by 63 ± 2 ( n = 15), 40 ± 4 ( n = 30), and31 ± 4% ( n = 16), respectively. Inhibition ofCaM resulted in a significant change in the EC 50 value forthe inhibition of ASP uptake by tetraethylammonium. Inconclusion, we demonstrate that the hOCT2 is inhibited by PI3K and PKAand activated by a CaM-dependent signaling pathway, probably via achange in substrate affinity. " @' V% B! c/ F9 S5 U2 F* b- f
      【关键词】 organic cation transport human organic cation transporter type human calcium/calmodulin calcium/calmodulindependent kinase II myosin light chain kinase phospholipase C protein kinase C proteinkinase A phosphatidylinositol kinase electrophysiology fluorescence microscopy [(dimethylamino)styryl] N met) O" T0 W  X" q8 b! n0 Y: `
   INTRODUCTION* D& S, f0 A/ X0 |9 \9 _8 g$ ^6 o9 g

THE TERM ORGANIC CATIONS REFERS to a broad class of compounds of endogenous (e.g.,choline, N 1 -methyl-nicotineamide, monoamineneurotransmitters such as dopamine and noradrenaline) and exogenous(e.g., cytostatics, antibiotics, opiates, antihypertensives,antiarrythmics, antihistamines, and sedatives) origin. The kidney isone of the most important organs in maintaining the homeostasis oforganic cations ( 16, 17, 46 ). Besides substrate-specific,Na -dependent organic cation transporters,Na -independent polyspecific organic cation transporters(OCTs) have been postulated for the epithelia in the intestine, liver,kidney, and also for the brain ( 15-17, 46 )." u- O( M1 S! T, \$ ]

In 1994, the first organic cation transporter from the rat (rOCT1) wascloned by expression cloning ( 6 ). rOCT1 was shown to bethe electrogenic organic cation transporter in the basolateral plasmamembrane of kidney and liver ( 2, 6, 14, 21, 34 ). Thesuccessive identification of several OCTs of different species byhomology cloning techniques ( 5 ) allowed ( 15-17, 19, 24, 36, 46-48 ) the investigation of molecular andphysiological properties of single OCTs. A common feature of the OCTsis the presence of several potential protein kinase phosphorylationsites in the intracellular loops, suggesting that their activitycan be subjected to regulation. We could indeed show that activation ofPKC with subsequent phosphorylation of a serine residue of rOCT1resulted in a stimulation of organic cation transport with an increase in substrate affinity and that rOCT1-mediated organic cation transport was also activated by PKA and endogenously stimulated by tyrosine kinases ( 21 ). Whereas rOCT1 is the principal OCT in therat kidney, hOCT2 has been suggested to be the most important one inthe human kidney ( 5 ). The more sensitive RT-PCR analysis showed additional transcription of hOCT2 mRNA in intestine, brain, spleen, and placenta ( 5 ). We also localized hOCT2 byRT-PCR in human isolated proximal tubules, and our functional studies with this preparation showed that hOCT2 is localized in the basolateral membrane ( 25 ). These results were recently confirmed byreal-time PCR and immunohistochemical analysis of the human proximaltubule ( 22 ).

Functional characterization of hOCT2 expressed in HEK-293 cells or Xenopus laevis oocytes with tracer flux measurements and patch-clamp studies showed that hOCT2, like other transporters of theOCT type (OCT1, OCT2 and OCT3), mediates electrogenic transport of small organic cations such as choline, tetraethylammonium(TEA ), N 1 -methyl-nicotineamide(NMN ), and 1-methyl-4-phenylpyridinium (MPP ).hOCT2 and other OCT-type transporters differ in their substrate specifities and affinities ( 1, 5, 15, 17, 46 ).1 k/ H5 M9 [+ H0 ^
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Regulation of organic cation transport is important for the secretionof multiple cationic endogenous substances, drugs, and otherxenobiotics and might explain the differences reported so far insubstrate affinities and differences in pharmacokinetics of organiccations. To date, only a few studies have addressed the regulation oforganic cation transport and none that of hOCT2. Regulation of thehuman extraneuronal monoamine transporter hEMT (also named hOCT3) byphosphorylation/dephosphorylation mechanisms was recently shown( 20 ). hEMT was inactivated by phosphorylation, especiallyby inhibition of MAP kinases, Ca 2  /calmodulin (CaM),phosphodiesterases, or protein phosphatases.

The purpose of the present study was to investigate the properties ofhOCT2 expressed in HEK-293 cells, with special emphasis on itsregulation by protein kinases.% s  u% U1 C* T; W9 n
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METHODS

HEK-293 cell culture. hOCT2 was stably expressed in HEK-293 cells (human embryonic kidneycortex cells, CRL-1573; American Type Culture Collection, Rockville,MD). Cells were grown at 37°C in 50-ml cell culture flasks (Greiner,Frickenhausen, Germany) in Dulbecco's modified Eagle's medium(Biochrom, Berlin, Germany) containing 3.7 g/l NaHCO 3, 1.0 g/l D -glucose, and 2 mM L -glutamine (GIBCOBRL/Life Technologies, Eggenstein, Germany) gassed with 8%CO 2. To this medium, 100,000 U/l penicillin, 100 mg/lstreptomycin (Biochrom), 10% fetal calf serum, and 0.8 mg/ml geneticin(GIBCO BRL/Life Technologies) were added. Experiments were performedafter 4-8 days with cells grown on glass coverslips from passages 11-42. Culture and functional analysisof these cells were approved by the LandesumweltamtNordrhein-Westfalen, Essen, Germany (521.-M-1.14/00).% v4 Z2 |  \3 ?  h( f5 h" X$ f
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Experimental solutions. As the superfusion solution, a HCO 3 − -free, Ringer-likesolution was used containing (in mM) 145 NaCl, 1.6 K 2 HPO 4, 0.4 KH 2 PO 4, 5 D -glucose, 1 MgCl 2, and 1.3 calcium gluconate,pH adjusted to 7.4. In some experiments, the superfusion solution wasmodified to (in mM) 95 NaCl, 50 KCl, 1.6 Na 2 HPO 4, and 0.4 NaH 2 PO 4 or 0 NaCl, 145 KCl, 1.6 Na 2 HPO 4, and 0.4 NaH 2 PO 4, with all other components kept constant." Z" _4 |- D% F
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Fluorescence measurements with4-[4-(dimethylamino)styryl]-N-methylpyridinium. As a substrate for hOCT2, the fluorescent organic cation4-[4-(dimethylamino)styryl]- N -methylpyridinium(ASP ) was used ( 26, 29, 39 ). The fluorescencemeasurement device and experimental procedure were as customary in ourlaboratory ( 13, 21, 32, 33 ). In short, measurements wereperformed in the dark with an inverted microscope (Axiovert 135, Zeiss, Oberkochen, Germany) equipped with a ×100 oil-immersion objective. Cells were excited pulsatory at 450-490 nm, and emission wasmeasured at 575-640 nm with a photon-counting tube (Hamamatsu H3460-04, Herrsching, Germany).

After addition of ASP  , fluorescence increased rapidly dueto cellular accumulation of ASP via hOCT2 (Fig. 1 ). As transport parameters, we evaluatedthe initial linear slope of the first 10-30 s (Fig. 1 ). In theinitial experiments examining the concentration dependence of anASP -induced fluorescence increase, we analyzed in additionthe maximal fluorescence reached. While the initial slope directlyrepresents the ASP uptake across the plasma membrane viahOCT2, the maximal fluorescence is the sum of ASP uptakeinto the cells, exit of ASP from the cells, intracellularcompartmentalization with changes in the emission spectrum, andbleaching of the dye ( 21, 25, 32, 33 ). In all experimentswith other substrates and regulators, only the initial slope of thecurves was determined as a transport parameter. To study regulation ofASP uptake, monolayers were incubated for 10 min with therespective agonists or inhibitors before ASP was added inthe continued presence of these substances.# `8 j/ N8 [. c) e. V: x+ e

Fig. 1. Concentration dependence of the uptake of4-[4-(dimethylamino)styryl]- N -methylpyridinium(ASP ) by HEK-293 cells expressing human organic cationtransporter type 2 (hOCT2). Values are means ± SE of initialfluorescence increase (10-30 s) and maximal fluorescence, with theno. of observations in parentheses. Inset : 2 originalrecordings of fluorescence increase by nontransfected andhOCT2-expressing HEK-293 cells after addition of ASP (1 µM) to the bath solution.

Fluorescence measurements with fura 2. Intracellular Ca 2  concentrations([Ca 2  ] i ) of HEK-293 cells were measured withthe Ca 2  -sensitive dye fura 2, as described previously( 31 ). Cells were incubated with the acetomethylester (fura 2-AM; 5 µM) dissolved with 0.1 g/l pluronic F-127 instandard solution for 40 min at 37°C in the dark. Measurements weredone with the same fluorescence system as described above (excitation:340, 360, and 380 nm; emission: 500-530 nm). The ratio of theemissions after excitations at 340 and 380 nm was calculated.Calibration of [Ca 2  ] i was done by incubatingthe cells with the Ca 2  ionophore ionomycin (1 µM) in thepresence (1.3 mmol/l) and nominal absence of Ca 2  with 5 mMEGTA present, according to standard methods ( 8 ).

Patch-clamp experiments. Membrane voltages ( V m ) were measured with theslow whole cell patch-clamp technique as performed before for rOCT1( 21 ). Pipettes were filled with a solution containing (inmM) 95 K gluconate, 30 KCl, 4.8 Na 2 HPO 4, 1.2 NaH 2 PO 4, 5 D -glucose, 0.73 Ca 2  gluconate, 1 EGTA, 1.03 MgCl 2, and 1 ATP, pH adjusted to 7.2. To this solution, 162 µM nystatin was added before use to permeabilize the membrane patchunder the pipette. Patch pipettes had an input resistance of ~10M. V m was measured in the current-clamp mode of a patch-clamp amplifier (U. Fröbe, Physiologisches Institut, Universität Freiburg, Freiburg, Germany).9 v: J/ ]! @" S

Chemicals. BAPTA, forskolin, sn -1,2-dioctanoyl glycerol (DOG),calphostin C,1-[ N, O -bis-(5-isoquinolinesulfonyl)- N -methyl- L -tyrosyl]-4-phenylpiperazine (KN-62),2-[ N -(4-methoxybenzenesulfonyl)]amino- N -(4-chlorocinnamyl)- N - methylbenzylaminephosphate (KN-92), calmidazolium,1-(5-iodonapthalene-1-sulfonyl)homopiperazine (ML-7),[1,4- diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] (UO126),{1-[6-((17 -3-methoxyestra-1,3,5(10)-trien-17-yl)- amino)hexyl]-2,5-pyrrolidinedione} (U-73122), wortmannin, and pluronic were purchased from Calbiochem (BadSoden, Germany). Human atrial natriuretic peptide was kindly provided by Dr. Knut Adermann (Niedersächsisches Institutfür Peptid-Forschung, Hannover, Germany). ASP waspurchased from Molecular Probes (Leiden, The Netherlands). All othersubstances and standard chemicals were obtained from Sigma(Taufkirchen, Germany) or Merck (Darmstadt, Germany)." e) E. J3 @/ ?1 f$ A! R
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Statistical analysis. Data are presented as originals or means ± SE, with n referring to the number of monolayers (fluorescence measurements) or cells (patch-clamp experiments). EC 50 values were obtainedby sigmoidal curve fittings (GraphPad InStat, San Diego, CA). Unpaired (fluorescence measurements) or paired (patch-clamp experiments) two-tailed Student's t -test was used to prove statisticalsignificance of the effects. Entire concentration-responsecurves were compared using an F -test. A P value significant.: I5 D& v4 k+ d: }( @: y. ~

RESULTS4 |2 G- f( S1 t/ O) y

Concentration dependence of ASP uptake. First, we determined the concentration dependence of ASP uptake by HEK-293 cells via hOCT2. At ASP concentrationsbetween 0.1 µM and 1 mM, the cellular fluorescence increasedconcentration dependently (Fig. 1 ). Saturation of the fluorescencesignal was only reached for the maximal fluorescence but not for theinitial rate. Concentrations of ASP higher than 1 mM couldnot be dissolved in aqueous solutions. Because 1 µM ASP already resulted in a significant cellular fluorescence increase (Fig. 1 ), all further fluorescence experiments were performed with thisASP concentration. Figure 1 also demonstrates thatASP uptake was mediated by hOCT2 because thenontransfected HEK-293 cells showed only a very small and slowfluorescence increase with time.

Substrate specificity. Next, we investigated the substrate specificity of hOCT2 by studyinginhibition of ASP uptake by known substrates of organiccation transport, like TEA  , TPA  , andcimetidine, and by the inhibitor of organic cation transporters, quinine ( 23 ). TPA  , TEA  , andcimetidine inhibited ASP uptake concentration dependently,with EC 50 values of 2.7, 35, and 36 µM, respectively(Fig. 2 ). ASP uptake wasalso concentration dependently inhibited by quinine, with anEC 50 value of 6.7 µM (Fig. 2 ).
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Fig. 2. Concentration-response curves for the inhibition ofinitial ASP uptake (1 µM) by the organic cationscimetidine, tetraethylammonium (TEA ), TPA  ,and quinine. Values are means ± SE expressed as%ASP uptake in the absence of these organic cations.EC 50 values determined from these curves were 36 (cimetidine), 35 (TEA ), 6.7 (quinine), and 2.7 µM(TPA ).

Electrogeneity of ASP uptake. Previous investigations showed the electrogeneity of hOCT2( 5 ), a known feature of the OCT-type transporters( 2, 6, 15, 21, 46 ). We confirmed this property of hOCT2 intwo ways. First, we measured the cellular fluorescence increase via hOCT2 by raising the K concentration in the superfusionsolution from 3.6 to 50 and 145 mM. Under these conditions, themembrane voltage of transfected HEK-293 cells depolarized from 44 ± 2 to 17 ± 1 and to  6 ± 1 mV( n = 6) ( 21 ). Initial ASP uptake was inhibited by 22 ± 8 ( n = 8) and55 ± 4% ( n = 9), respectively (Fig. 3, left ). Second, we proved theelectrogeneity of hOCT2 by determining the dependence of membranedepolarization on the ASP concentration in the bath withthe slow whole cell patch-clamp technique. Increasing ASP concentrations from 1 µM to 1 mM resulted in increasing membrane depolarizations (Fig. 3, right ).

Fig. 3. Electrogeneity of ASP uptake by hOCT2. Left : decrease in initial ASP (1 µM)fluorescence increase when cells were depolarized by increasing theextracellular K concentration from 3.6 to 50 or 145 mM. Right : change in membrane voltages( V m ) of HEK-293 cells expressing hOCT2 inducedby addition of ASP to the bath solution at concentrationsbetween 1 and 1,000 µM. Values are means ± SE, with the no. ofobservations in parentheses. All effects are significant( P
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Regulation by PLC and PKC. To investigate the involvement of the PLC and the subsequent PKCsignaling pathway in the regulation of hOCT2, we tested different agonists. Incubation of cells with the muscarinic agonist carbachol resulted in a concentration-dependent inhibition of ASP uptake at concentrations between 0.1 and 100 µM (Fig. 4 ). Maximal inhibition at 100 µMcarbachol was 44 ± 3% ( n = 14). Withoutincubation, this carbachol concentration had no significant effect onASP uptake ( n = 10), indicating that thepositively charged carbachol did not interfere significantly with hOCT2as substrate.( }& T8 ]$ z* N; o

Fig. 4. Concentration-response curves for the effects ofcarbachol, ATP, and forskolin on initial ASP uptake (1 µM). Values are means ± SE, with the no. of observations inparentheses.
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As another activator of the PLC signaling pathway, we used ATP, whichactivates PLC through purinergic receptors. ATP led to aconcentration-dependent inhibition of ASP transport atconcentrations between 0.1 and 100 µM. The maximal ATP concentrationof 100 µM led to an inhibition of ASP transport by34 ± 4% ( n = 21) (Fig. 4 ).

To examine whether the effect of carbachol on ASP uptakewas mediated via PKC, we incubated cells with the PKC activator DOG, which is a membrane-permeable analog of diacylglycerol. DOG (1 µM) had no significant effect on ASP transport ( 3 ± 5%, n = 26) (Fig. 5 ).Thus involvement of PKC in organic cation transport via hOCT2 isunlikely. We verified this conclusion by simultaneous incubation of thecells with carbachol and either one of the PKC inhibitors calphostin C(0.1 µM, n = 12) or tamoxifen (20 µM, n = 5). There were no significant differences betweenthe effects of carbachol in the absence and presence of these PKCinhibitors (Fig. 5 ). We also excluded endogenous activation of PKC byincubating the cells with calphostin C alone ( n = 6), which again had no significant effect on ASP transport(Fig. 5 ).

Fig. 5. Effects of PKC [ sn -1,2-dioctanoyl glycerol(DOG); 1 µM] and PLC (carbachol; 100 µM) activation on initialASP uptake (1 µM). Calphostin C (0.1 µM) or tamoxifen(20 µM) was used as a PKC inhibitor. Values are means ± SE,with the no. of observations in parentheses. * Statisticallysignificant effect ( P7 L( Z0 k# w6 `5 c  r
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Regulation by PKA. To investigate the involvement of the cAMP-dependent protein kinase(PKA) in the regulation of organic cation transport via hOCT2, we usedthe adenylate cyclase activator forskolin. Compared with carbachol orATP, forskolin had a weaker concentration-dependent effect onASP uptake (Fig. 4 ). At 1 µM forskolin, ASP uptake was significantly reduced by 18 ± 4% ( n = 40).
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Regulation by Ca 2 and theCa 2  /CaM complex. Because activation of G protein-coupled receptors by carbachol or ATPreduced ASP uptake most likely independently of PKC, wefurther investigated whether this effect involves an increase incytosolic Ca 2  and the Ca 2  /CaM complex. Toverify an increase in [Ca 2  ] i after carbacholor ATP in HEK-293 cells and to estimate its magnitude, Ca 2  measurements with fura 2 were conducted. Carbachol (100 µM) and ATP(100 µM) increased [Ca 2  ] i by 300 ± 40 ( n = 5) and 420 ± 62 nM ( n = 5), respectively; therefore, the role of[Ca 2  ] i in the regulation of hOCT2 wasexamined. Reduction of the extracellular Ca 2  concentrationfrom 1.3 mM to 1 µM Ca 2  resulted in an inhibition ofASP transport by 50 ± 5% ( n = 16)(Fig. 6 ). Reduction of[Ca 2  ] i by incubation of cells with theCa 2  chelator BAPTA (5 µM) led to a reduction inASP uptake by 41 ± 5% ( n = 15)(Fig. 6 ). The incubation of cells with carbachol in the presence ofBAPTA resulted in a significantly, even larger, reduction inASP uptake compared with either carbachol or BAPTA alone,indicating an independence of the carbachol effect of[Ca 2  ] i (Fig. 6 ).9 o$ g# T9 D6 X9 O: U
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Fig. 6. Effects of inhibition of calmodulin (CaM; calmidazolium;5 µM), PLC activation (carbachol; 100 µM), lowering ofintracellular Ca 2  concentration (low Ca 2  :extracellular Ca 2  = 1 µM), or chelation ofintracellular Ca 2  (BAPTA; 5 µM) on initialASP uptake (1 µM). The independence of the effects ofcarbachol (100 µM) and calmidazolium (5 µM) or BAPTA (5 µM) isalso demonstrated. Values are means ± SE, with the no. ofobservations in parentheses. * Statistically significant effect( P

Inhibition of CaM with the CaM inhibitor calmidazolium (5 µM) reducedASP transport by 63 ± 2% ( n = 15)(Fig. 6 ), indicating a significant endogenous activation of hOCT2 viathis complex. To exclude a possible interference of the cationiccalmidazolium with hOCT2, the effects of 1, 5, and 50 µMcalmidazolium were tested acutely without incubation. Inhibition ofASP uptake was 65 ± 4 ( n = 10),64 ± 3 ( n = 18), and 63 ± 5%( n = 6), respectively. When CaM was inhibited bycalmidazolium (5 µM) and the cells were stimulated simultaneouslywith carbachol (100 µM), ASP uptake was moresignificantly reduced than with either carbachol or calmidazolium alone(28 ± 2%, n = 16) (Fig. 6 ). This suggests thatat least part of the carbachol effect is mediated via aCa 2  /CaM-independent pathway. To test whether activation ofPLC is involved in the carbachol-mediated inhibition ofASP uptake, carbachol (100 µM) was added in the presenceof the PLC inhibitor U-73122 (5 µM). Under these conditions,carbachol still reduced ASP uptake by 55 ± 11%( n = 18) (Fig. 7 ).U-73122 per se did not significantly inhibit basal transport activity( 10 ± 7%, n = 17) (Fig. 7 ). To identify thekinases that are involved in the Ca 2  /CaM-mediatedregulation of hOCT2, we examined ASP uptake afterincubation of cells with KN-62, an inhibitor of the multifunctionalCa 2  /CaM-dependent protein kinase II (CaMKII). KN-62 (1 µM) inhibited ASP transport significantly by 40 ± 4% ( n = 30) (Fig. 8 ).The inactive analog KN-92 (1 µM) had no statistically significanteffect on ASP uptake ( 4 ± 6%, n = 21) (Fig. 8 ), indicating the specificity of the effect of KN-62.Because the Ca 2  /CaM complex also activates the myosinlight chain kinase (MLCK), we tested the effect of the MLCK inhibitorML-7. Incubation with ML-7 (3 µM) also led to an inhibition ofASP transport by 31 ± 4% ( n = 16)(Fig. 8 ). These findings suggest that CaMKII and MLCK are endogenouslyactivated in HEK-293 cells and stimulate ASP uptake viahOCT2.
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Fig. 7. Effect of inhibition of phosphatidylinositol 3-kinase bywortmannin (0.1 µM) on carbachol-mediated (100 µM) transportreduction and independence of carbachol-mediated (100 µM) transportreduction of PLC inhibition by U-73122 (5 µM). Values are means ± SE, with the no. of observations in parentheses. The effects ofcarbachol in the absence or presence of U-73122 are not significantlydifferent.* Statistically significant effect ( P3 t( L* i2 p0 }0 ~$ M9 v6 `

Fig. 8. Effects of inhibition of Ca 2  /CaM-dependentkinase II (CaMKII) by1-[ N, O -bis-(5-isoquinolinesulfonyl)- N -methyl- L -tyrosyl]- 4-phenylpiperazine(KN-62; 1 µM) and its inactive analog2-[ N -(4-methoxybenzenesulfonyl)]amino- N -(4-chlorocinnamyl)- N -methylbenzylaminephosphate (KN-92; 1 µM) or of myosin light chain kinase by1-(5-iodonapthalene-1-sulfonyl)homopiperazine (ML-7; 3 µM) on initialASP uptake (1 µM). The independence of the effects ofcarbachol (100 µM) and of KN-62 (1 µM) is also demonstrated. Valuesare means ± SE, with the no. of observations in parentheses.* Statistically significant effect ( P

To examine whether the effect of carbachol on hOCT2 is independent notonly of Ca 2  /CaM but also of CaMKII, cells weresimultaneously incubated with carbachol (100 µM) and KN-62 (1 µM).This resulted in an inhibition of ASP transport by 64 ± 3% ( n = 12) (Fig. 8 ), which was significantly greater than the effect of carbachol or KN-62 alone, indicating theadditivity of these two signaling pathways.
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Regulation by the phosphatidylinositol 3-kinase. Because activation of G protein-coupled receptors can also stimulatephosphatidylinositol 3-kinase (PI3K), we tested whether the effect ofcarbachol is influenced by inhibition of PI3K. Incubation with the PI3Kinhibitor wortmannin (0.1 µM) alone significantly stimulated basaltransport activity of HEK-293 cells (55 ± 17%, n = 16) (Fig. 7 ), indicating endogenous activation of this enzyme. Pretreatment of the cells with wortmannin could almost completely prevent the effect of simultaneous incubation with carbachol (100 µM)on initial ASP uptake ( 7 ± 5%, n = 11) (Fig. 7 ). Further downstream in the signaling cascade are alsothe mitogen-activated kinase kinases MEK1 and MEK2. Inhibition of thesekinases by UO126 (1 µM) had no effect on basal ASP transport ( 4 ± 12%, n = 6) or oncarbachol-induced inhibition of ASP uptake ( 42 ± 4%, n = 6).& d& J8 a& o2 {9 x

Influence of protein kinase activation on substrate affinities. For the OCT1 of rat (rOCT1), we reported that PKC activation results ina modulation of the affinities for various substrates ( 21 ). To examine whether inhibition of the hOCT2 byblocking the Ca 2  /CaM complex or by carbachol also resultsin altered substrate affinities, EC 50 values forTEA were determined under these conditions and comparedwith those obtained under control conditions. Activation of the Gprotein-coupled receptor signaling pathways by carbachol (100 µM) orinhibition of the CaMKII by KN-62 or of PI3K by wortmannin induced nosignificant changes in the concentration-response curves for theinhibition of ASP uptake by TEA (Fig. 9 ). Conversely, inhibition of CaM by theCaM antagonist calmidazolium (5 µM) significantly reduced theapparent affinity of hOCT2 for TEA (EC 50 = 249 µM) compared with 35 µM under basal conditions./ m; u; M) }* N4 B5 F) w8 U

Fig. 9. Concentration-response curves for the inhibition ofinitial ASP uptake (1 µM) by TEA in theabsence or presence of carbachol (CCH; 100 µM), KN-62 (1 µM),wortmannin (0.1 µM), or calmidazolium (5 µM). Only the curve forcalmidazolium differs significantly from the control curve. TheEC 50 value increased from 35 (control) to 249 µM(calmidazolium). Values are means ± SE, with the no. ofobservations in parentheses.$ e, Y* s1 B+ u1 ], B
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DISCUSSION
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Organic cation transport in epithelia is mediated by severalpolyspecific transporters, which initially have been examined functionally using different experimental models and techniques ( 15-17, 46 ). In the renal proximal tubule, three maintransport systems for organic cations were functionally differentiated: a basolaterally localized electrogenic uniporter, a luminally localizedelectroneutral H /cation exchanger, and a luminallylocalized electrogenic uniporter with high affinity to choline( 17 ). The cloning of the first organic cation transporterfrom the rat (rOCT1) in 1994 ( 6 ) made it possible tospecifically characterize single organic cation transporters afterstable transfection in different cell models ( 15, 16, 21, 46 ). Functional and morphological studies suggested that thetransporters of the OCT type are polyspecific organic cationtransporters localized in the basolateral membrane ( 2, 6, 14, 21, 23, 34 ). In 1997, the first human organic cation transporterswere cloned ( 5, 7, 35, 43, 47 ).! _7 w% e6 a8 o4 W8 z9 ?! x

In this study, we investigated the properties and regulation of hOCT2stably transfected in a human embryonic kidney cell line (HEK-293).Dynamic microfluorimetry with the fluorescent organic cationASP  , as adapted in our laboratory ( 13, 21, 25, 32, 33 ), and the slow whole cell patch-clamp technique were used. Weconfirm the electrogeneity of this transporter and describe itssubstrate specificity for some classic organic cations. Mostimportantly, we demonstrate for the first time that hOCT2 is regulatedby various intracellular signaling pathways. It is inhibited by PKA,PLC, and PI3K activation but not by PKC activation. It is endogenously activated by the Ca 2  /CaM complex and theCa 2  /CaM-dependent kinases CaMKII and MLCK.. @* a* J8 ~+ z( g) i7 j7 j# `9 T
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The fluorescent organic cation ASP is taken upconcentration dependently via hOCT2. However, as shown before for rOCT1( 21 ), the initial rate of cellular ASP accumulation did not reach saturation in the range of concentrations used. As discussed before ( 33 ), this may indicate that athigher concentrations ASP is accumulated in intracellularcompartments. While the initial slope of the uptake, analyzed duringthe first 10-30 s, directly represented ASP uptakeacross the plasma membrane of HEK-293 cells, the fluorescence maximumreflected a steady state of ASP uptake into the cells,ASP transport out of the cells, intracellularcompartmentalization, and bleaching of the dye ( 21, 25 ).Therefore, transport was analyzed as the initial rate of fluorescenceincrease, using low concentrations of ASP (1 µM). Asshown in Fig. 1, initial uptake of ASP by thesetransfected HEK-293 cells was mediated by hOCT2 only.
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Substrate specificity. Previous radioactive tracer or electrophysiological studies on hOCT2expressed in HEK-293 cells or X. laevis oocytes showed thathOCT2 transports small organic cations, such as TEA  ,TPA  , NMN  , and choline, and isnoncompetitively inhibited by larger organic cations, such as quinine, d -tubocurarine, and vecuronium ( 15-17, 42, 46 ). The EC 50 values obtained for the inhibition ofASP uptake in the present study by TEA (EC 50 = 35 µM), TPA (EC 50 = 2.7 µM), cimetidine (EC 50 = 36 µM), and quinine (EC 50 = 6.7 µM) are similar to K m or K i valuesdetermined with radiolabeled substrates for hOCT2 expressed in X. laevis oocytes: K m for radiolabeled TEA was 76 µM, and K i for theinhibition of radiolabeled TEA uptake by TPA or quinine were 1.5 and 3.4 µM, respectively ( 5 ).. v, ]  z0 n0 Y; m7 S

Electrogeneity of hOCT2. A common feature of all OCT-type transporters is their electrogeneity( 15-17, 46 ). In the present study, we confirmed this electrogeneity in two ways: 1 ) in microfluorimetric studies,ASP uptake was dependent on membrane voltage and decreasedwith membrane depolarization induced by increases in extracellularK concentration (Fig. 3 ); and 2 ) inelectrophysiological studies, increasing ASP concentrations led to increasing depolarization of the membrane voltageof transfected HEK-293 cells due to the influx of positive charges.While the first finding could also be due to changes in substratebinding, the second clearly indicates electrogenic transport.

Regulation of hOCT2. The regulation of organic cation transport has been under investigationonly recently ( 13, 21, 25 ). Organic cation transport across the basolateral membrane of proximal tubules was activated inrabbits ( 12 ) and inhibited in humans ( 25 ) byPKC stimulation. An increase in transport mediated by the rOCT1 wasshown by us for PKC and PKA and the tyrosine kinasep56 lck ( 21 ). Similar differences inregulation were also demonstrated by us for organic cation transportacross the apical membrane of human and porcine proximal tubule celllines ( 13 ). These findings suggest species- and alsosubtype-specific differences in transporter regulation. For OCT2 aswell as for OCT1 transporters from rats and humans, several potentialphosphorylation sites for PKA, PKC, and tyrosine kinases weredescribed; some of them are conserved through all four isoforms( 5, 6 ).3 C5 ^$ c/ q5 D! G2 O% C" A. o% y
$ g6 u; W$ B1 Z4 i+ O5 ~
cAMP-dependent PKA. Addition of the adenylate cyclase activator forskolin leading toactivation of PKA resulted in a small concentration-dependent inhibition of ASP uptake via hOCT2. Recently, wedemonstrated that in freshly isolated human proximal tubules organiccation transport across the basolateral membrane, probably mainlymediated via hOCT2, is also downregulated by PKA to a similar extent( 25 ). hEMT (hOCT3) again expressed in HEK-293 cells showedno cAMP-dependent regulation, and only nonspecific high concentrationsof forskolin (250 µM) led to a small reduction in radiolabeledMPP transport in that study ( 20 ).
, C' R5 l+ U( E6 k% ^( ^3 w
G protein-coupled receptor and PKC. To investigate the effect of activation of G protein-coupled receptoron ASP uptake in hOCT2-transfected HEK-293 cells, we usedthe purinergic agonist ATP and the muscarinic agonist carbachol.Receptors for both agonists have been demonstrated in HEK-293 cells( 27, 37 ). ATP and carbachol resulted in inhibition ofASP uptake by ~34 and 44%, respectively. We excludedthe involvement of PKC in this effect by using the direct PKC activatorDOG, a membrane-permeable analog of diacylglycerol. DOG had nosignificant effect on hOCT2-mediated ASP uptake. The factthat PKC was not involved in the effect of carbachol was furtherverified by simultaneous incubation of the cells with carbachol and thePKC inhibitors calphostin C or tamoxifen. Neither PKC inhibitorsignificantly influenced the carbachol-induced inhibition ofASP transport. This absence of a PKC-mediated regulationof hOCT2, therefore, parallels similar observations for hOCT3( 20 ) but is in contrast to the PKC-mediated activation ofrOCT1 ( 21 ) or organic cation transport across thebasolateral membrane of isolated rabbit proximal tubules (PKC-mediatedactivation) ( 12 ) or isolated human proximal tubules(PKC-mediated inhibition) ( 25 ). These findings alsounderline the marked differences in transport properties of theseorganic cation transporters between the different species and,possibly, the expression systems. Furthermore, it should be kept inmind that for hOCT1 and hOCT2 various splice variants were identifiedand that, especially in the human proximal tubule, expression of asplice variant to the originally cloned hOCT2 as the relevanttransporter was described recently ( 11, 40 ).$ l. R6 b# r/ L* a4 f! ?" x
% f1 Q$ N( v7 L6 ?
CaM and Ca 2 /CaM-dependent kinases. After exclusion of PKC involvement in the regulation of hOCT2, wefurther investigated whether CaM and CaMKII or MLCK is involved in theregulation of hOCT2-mediated ASP uptake. Inhibition of CaMwith calmidazolium resulted in a marked reduction in hOCT2 transport by63%, indicating an endogenous activation of the Ca 2  /CaMpathway. In line with this effect of CaM inhibition is the reducedactivity of hOCT2 when cellular Ca 2  was decreased by usingthe Ca 2  chelator BAPTA or by lowering extracellularCa 2  to 1 µM, which also leads to reduction in cellularCa 2  levels. Activation of CaM by Ca 2  stimulates the multifunctional CaMKII, which is known to phosphorylate many target proteins, leading to changes in their activity.Consequently, also an inhibition of CaMKII by KN-62 reducedhOCT2-mediated ASP transport by 40%. We verified thespecificity of this inhibition by KN-62 with the inactive analog KN-92,which was without a significant effect. Because KN-62 inhibits not onlyCaMKII but also CaMKIV and CaMKV with similar affinities, these kinasesmay be involved in the observed effects as well. In addition to CaMKII,MLCK is also activated by CaM. Inhibition of MLCK by ML-7 reducedASP uptake via hOCT2 again by 31%. Together these dataindicate that hOCT2 is endogenously activated via CaM-dependent CaMKIIand MLCK stimulation. A similar regulation was shown for the renalbasolateral PAH transporter by CaMKII in isolated proximal tubules ofthe rabbit ( 3 ). The involvement of CaMKII was alsodemonstrated for the regulation of the Na /H exchanger ( 41 ), the Na -HCO 3 − cotransporter, or proline uptake in the proximal tubule ( 30, 44, 45 ). A CaM-dependent regulation of organic cation transport wasonly described for hOCT3 ( 20 ) but has not been examinedfor any other cloned OCT so far.
9 w+ `! l8 ]8 c! c9 J$ G9 W- Y
Simultaneous activation of the G protein-coupled receptor signalingpathway by carbachol and inhibition of either CaM by calmidazolium orCaMKII by KN-62 had additive effects on ASP uptake,indicating the independence of the carbachol-mediated inhibition andthe CaM-mediated stimulation of hOCT2 transport. Carbachol or ATPincreased [Ca 2  ] i in this and previousreports from HEK-293 cells ( 27, 37 ). This increase in[Ca 2  ] i should have increased CaM activityand, therefore, stimulated hOCT2 transport; however, the opposite wastrue. This would indicate an effect of carbachol or ATP independentlyof Ca 2  and overriding the stimulation of CaM. This wasfurther supported by the increased inhibition of hOCT2-mediatedASP uptake when the G protein-coupled receptor signalingpathway was activated by carbachol in the presence of BAPTA comparedwith either one alone. Additional evidence for the independence of thecarbachol inhibition of ASP uptake from the PLC-activatedsignaling pathway derives from experiments performed under inhibitionof the agonist-induced PLC activation with U-73122: inhibition ofASP uptake was still present even after simultaneousincubation of cells with carbachol and U-73122.
. {) v5 T! f6 ^% u7 S
The observed effects of carbachol and calmidazolium on ASP uptake by hOCT2 could be also explained by a direct interaction at thetransport binding site because these drugs are organic cations.However, for carbachol this can be excluded for the following reasons:acute administration of carbachol had no significant effect onASP uptake; published affinity of carbachol for thebasolateral organic cation transport system is 12.2 mM( 39 ); carbachol had no effect on ASP uptakeby hOCT1 or rOCT1 (Ciarimboli and Schlatter, unpublished observations); and carbachol incubation caused no change in the concentration-response curve for the inhibition of ASP uptake by TEA  . Acute addition of calmidazolium resulted inan inhibition of ASP uptake similar to that afterincubation. This fast effect within seconds is compatible with data forthis inhibitor in other systems ( 18 ). A significant directinteraction of calmidazolium with hOCT2, however, can again be excludedfor the following reasons: inhibition of uptake did not differ between1 and 50 µM calmidazolium; maximal inhibition of transport reachedonly ~60%, whereas other substrates inhibited hOCT2 by up to 100%;and calmidazolium incubation caused a rightward shift in theconcentration-response curve for the inhibition of ASP uptake by TEA  , whereas a leftward shift should have beenexpected if calmidazolium were to be a substrate for the transporter.Therefore, the observed inhibition of ASP uptake bycalmidazolium is due to regulation via CaM.% L8 P* Q* j" d( a. f9 S  p
& Z: X$ k, U2 Q4 _. R3 U
PI3K. Stimulation of G protein-coupled receptors mediates intracellularsignaling via GTP-bound G protein -subunits or -complexes. TheGTP-bound G protein -subunits initiate the classic cascade involvingdiacylglycerol and inositol 3,4,5-trisphosphate, whereas the -complexes activate a cascade that connects G receptor activation to PI3K and the subsequent MAPK signaling pathway ( 9, 10 ). We have therefore tested whether the signal pathway mediated by -complexes is involved in the regulation of hOCT2 by carbachol. The incubation of the cells with wortmannin, a potent inhibitor of PI3Kactivity, significantly stimulated the ASP uptake viahOCT2, showing that the enzyme is endogenously activated. Stimulationof the G protein-coupled receptor by carbachol and the contemporaneousinhibition of the PI3K activity by wortmannin almost completelyprevented the reduction of initial ASP uptake of HEK-293cells. The involvement of PI3K was also demonstrated for the regulationof renal organic anion transporter and of P-glycoprotein-mediated drugefflux. Wortmannin was able to inhibit tubular basolateral transport ofPAH in isolated proximal tubules from rabbit kidney ( 4 )and to inhibit apical P-glycoprotein-mediated drug efflux in theproximal tubule of mice ( 38 ). The involvement of Greceptor-linked stimulation of MAPK activity was also demonstrated forthe regulation of Na-HCO 3 − cotransport activity after muscarinic stimulation by carbachol in the opossum kidney proximal tubule cell line ( 28 ). The components of the signalingpathway of G protein-coupled receptors downstream of PI3K probably have no influence on hOCT2 activity, because inhibition of MEK1 and MEK2 byUO126 had no effect on basal ASP transport or oncarbachol-induced inhibition of ASP uptake.; t8 b$ g  c! G& w3 C4 l4 P

TEA affinity and hOCT2 regulation. Recently, we demonstrated the increase in affinity for the organiccation transporter rOCT1 by PKC-dependent phoshorylation ( 21 ). To find out whether the affinities of hOCT2 werealso changed when transport is downregulated, e.g., by activation of the G protein-coupled receptor pathway, or by CaM or CaMKII inhibition, we determined concentration-response curves for TEA  , whencells were incubated with carbachol, calmidazolium, KN-62, orwortmannin. Only incubation with calmidazolium resulted in asignificant change in the concentration-response curves for theinhibition of ASP uptake by TEA (Fig. 9 ).This is further evidence for the independence of the carbachol effecton transport activity from the CaM pathway. The absence of an effect ofKN-62 on the EC 50 value for TEA could beexplained by assuming that CaM acts directly on the transporter or bythe fact that inhibition by KN-62 is too low to detect changes in theEC 50 value in this system. These findings, together withthe finding that contemporaneous incubation with carbachol andwortmannin almost completely prevented the reduction of initialASP uptake observed with carbachol alone, suggest that thePI3K signaling pathway can be directly involved in the regulation ofhOCT2 after stimulation of the G protein-coupled receptor.% \+ z) {7 J/ g) d& v

In conclusion, we have shown that several signaling pathways areinvolved in the regulation of hOCT2-mediated organic cation transport.Whereas PKA stimulation resulted in inhibition, endogenously active CaMor the CaM-dependent kinases CaMKII and MLCK led to activation, andinhibition of this pathway led to a reduction, of hOCT2-mediatedtransport. Activation of PKC had no effect on this transporter.Activation of the G receptor-coupled signaling pathway by carbacholinhibited hOCT2-mediated transport, probably acting through PI3K.Regulation of organic cation transporters may alter the secretion andabsorption of multiple endogenous substances, drugs, and otherxenobiotics, leading to changes in pharmacokinetics of these organic cations.' p. E8 ]% l  B2 i
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ACKNOWLEDGEMENTS
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We thank Ute Kleffner, Ingrid Kleta and Ulrich Siegel for technical assistance.
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