Short-Term Regulation of Basolateral Organic Anion Uptake in Proximal Tubular Opossum Kidney Cells: Prostaglandin E2 Acts via Receptor-Mediated Activation of Protein Kinase A
Christoph Sauvant,
Hildegard Holzinger and
Michael Gekle
Physiologisches Institut der Universität Würzburg, Würzburg, Germany
Correspondence to Dr. Christoph Sauvant, Physiologisches Institut, Universität Würzburg, Röntgenring 9, 97070 Würzburg, Germany. Phone: ++49-931-31-2724; Fax: ++49-931-31-2741; E-mail: christoph.sauvant{at}mail.uniwuerzburg.de
ABSTRACT. It was shown previously that EGF induces release ofthe important prostanoid prostaglandin E2 (PGE2) in proximaltubular opossum kidney (OK) cells and PGE2 then stimulates initialbasolateral uptake of organic anions (OA) dose dependently.PGE2 is a receptor agonist and a known substrate for the basolateralexchanger mediating OA uptake (OAT1 and/or OAT3). This studyinvestigated the mechanism of short-term PGE2 action on initialbasolateral OA uptake in OK cells. PGE2 stimulation of OA uptakewas abolished by selective inhibition of adenylate cyclase (byMDL-12, 330A) or protein kinase A (PKA; by H89). PGE2 stimulationof OA uptake persisted after preloading the cells with glutarateand was still abolished by inhibition of PKA. Selective activationof adenylate cyclase by forskolin led to identical results.These data contradicted the hypothesis that PGE2 action on OAuptake is due to its action as a counter ion. Therefore, wetested whether the PGE2 receptors (EP1 to 4) are involved instimulation of OA uptake in OK cells by PGE2. Because of theirintracellular signaling profile, EP1 and EP3 were not takeninto account as possible receptors for mediation of PGE2-inducedOA uptake. With the use of selective agonists (11-deoxy PGE1and butaprost), EP4 was pharmacologically identified as thereceptor responsible for PGE2-mediated stimulation of OA uptake.By reverse transcription–PCR, cloning, and subsequentsequencing, a homologue fragment to EP4 was identified in OKcells. EGF-induced stimulation of basolateral organic anionuptake was abolished by inhibition of adenylate cyclase or PKA.This indicates that EGF action is mediated by generation ofPGE2. The following model is proposed: PGE2 generated in thecells does not act as a counter ion but activates adenylatecyclase. This is mediated by a homologue of EP4 receptor. cAMPthen activates PKA, which stimulates initial basolateral uptakeof OA in OK cells by a not-yet-known mechanism. PGE2 is an organicanion, a potential stimulator of organic anion excretion, andan important mediator of inflammation all at once. Thus, themechanism presented here may contribute to a limitation of inflammatoryevents in the kidney cortex interstitium.
The organic anion transport system of the renal proximal tubuleplays a crucial role in the excretion of a variety of potentiallytoxic compounds (1,2). This system consists of a basolaterallylocated, polyspecific organic anion (OA) exchanger and a lesswell-characterized transport step at the apical membrane (3,4).
The basolateral OA exchanger is a tertiary active transportsystem, dependent on an inward-directed Na+ gradient to drivethe uptake of -ketoglutarate, which is then exchanged for OA(2,3,5,6). The basolateral exchanger for OA and dicarboxylateswas cloned by three independent groups (7–9) in 1997 andnamed OAT1. The homologous protein was cloned from human kidneyand was called hOAT1 (10,11) or hPAHT (12). The genomic DNAfrom human hOAT1 is organized in 10 exons, and up to now, fourisoforms have been described (13). Furthermore, it was shownthat OAT1 represents characteristics of the basolateral, polyspecifictransporter for OA (14), which has been functionally describedfor a substantial amount of time (6). Meanwhile, there is evidencethat the basolaterally located OAT3 from rat also acts as anexchanger for dicarboxylates and OA as OAT1 does (15).
Furthermore, three additional homologues were cloned and namedhOAT2, hOAT4, and hOAT5 (16). HOAT4 is expressed in kidney andplacenta (17). Up to now, it seemed that hOAT5 was not expressedin the kidney (16). hOAT2 shows 37% homology in amino acid sequencecompared with hOAT1 (16) and differs from OAT1 in substratespecificity. There is also evidence that OAT2 is located inthe luminal membrane of the proximal tubular epithelium (18).Furthermore, these proteins are no exchangers like OAT1 or OAT3but seem to work as facilitators (19).
Little is yet known about the modulation of this transport system(2,3,20). In fact, most of the publications available up tonow investigated the effect of protein kinase C (PKC) in differentspecies, setups, or cell lines. Thereby, it was shown that stimulationof PKC (e.g., by parathyroid hormone, bradykinin, or phenylephrine)leads to an inhibition of basolateral OA transport (21–23).You et al. (24) showed that PKC inhibits murine OAT withoutdirect phosphorylation of the transport protein itself. As wecould show only recently, EGF stimulates the basolateral OAtransport via mitogen-activated protein kinases (MAPK) (25).MAPK then activate phospholipase A, generating arachidonic acid,which is converted to prostaglandin E2 (PGE2) by cyclooxygenase1 (COX1). PGE2 then dose-dependently stimulates basolateralOA uptake by a not-yet-known mechanism (26) (see Figure 1).
Figure 1. Summary indicating the main questions addressed in this article. Former studies indicated that EGF stimulates the PAH/dicarboxylate exchanger via activation of the mitogen-activated protein kinases (MAPK) MEK and ERK1/2 (23). ERK1/2 then activates phospholipase A2 (PLA2), increasing the release of arachidonic acid. Arachidonic acid is then metabolized into prostaglandin E2 (PGE2), which then stimulates initial organic anion (OA) uptake (24). This article investigates the mechanisms by which PGE2 stimulates basolateral OA uptake in proximal tubular opossum kidney (OK) cells. Does PGE2 act as countersubstrate (I) or via a receptor-mediated pathway (II)?
Prostanoids are important regulatory factors in the kidney.Renal tubular epithelia and interstitial cells synthesize andrelease predominantly PGE2 in a regulated manner. PGE2 affectsurine concentration ability; promotes salt and water concentrationability; promotes salt and water excretion by the renal medulla;maintains renal blood flow and GFR, particularly under neurohumoralactivation; and stimulates renin release (27). Furthermore,PGE2 is involved in inflammatory events in kidney tissue (28).PGE2 initiates and maintains its physiologic effects throughinteraction with specific receptors on target cell surface membranes.At least four different PGE2 receptor isoforms exist, designatedEP1 to 4, which all are expressed in the kidney (29,30). TheEP2 and EP4 subtypes act via activation of adenylate cyclase,whereas EP3 inhibits adenylate cyclase activity and stimulationof EP1 leads to activation of PKC. In addition, it is knownthat PGE2 is a substrate for the OA secretory transport system(31) and also may serve as substrate and/or counter ion forthe basolateral OA exchanger (which may consist of OAT1 and/orOAT3, as already mentioned) (32).
Thus, we investigated the mechanisms of PGE2-induced stimulationof basolateral OA uptake in more detail. For this stimulationto occur, two mechanisms are possible. First, PGE2 could actas a counter substrate and thus stimulate the uptake of OA.Second, PGE2 could act via coupling to specific receptors presentat the membrane of proximal tubular OK cells (see Figure 1).As a model system, we chose the proximal tubule–derivedOK cell line cultured on permeable supports, a functionallywell-characterized cell line to investigate basolateral OA transportvia the dicarboxylate exchanger mechanism (25,33). Meanwhile,the functionally predicted OAT1 from OK cells is partly cloned(Dr. Y. Hagos, Goettingen; GenBank AJ308236), showing 88% sequencehomology to human OAT1. In the following, we present data showingthat PGE2 stimulates basolateral OA uptake by binding a specificreceptor, which, via cAMP, stimulates protein kinase A (PKA).PKA then stimulates initial basolateral OA uptake.
Cell Culture
OK cells were obtained from Dr. Biber (Department of Physiology,University of Zurich, Zurich, Switzerland). Cells were maintainedin culture at 37°C in a humidified 5% CO2, 95% air atmosphere.The growth medium was MEM (pH 7.4), supplemented with Earl’ssalts, nonessential amino acids, 10% (vol/vol) FCS (BiochromKG, Berlin, Germany), and 26 mmol/L NaHCO3. Cells were culturedon permeable supports (3-µm pore diameter; Falcon, BectonDickinson Labware, Franklin Lakes) for transport measurements.The effective growth area on one permeable support was 4.3 cm2/filter.All studies were performed between passages 60 and 100. Theseeding density was 0.4 · 106 cm-2. The medium was changedevery third day, and the monolayers were used for experimentsat day 10 after seeding. All experiments were performed withcells that were serum-starved for 24 h before the experiments.
Transport Measurements General Setup.
The volumes of the apical and basolateral compartment were 1.3ml and 2.5 ml to avoid hydrostatic pressure differences. Beforeeach experiment, the cells were washed three times with PBS(138 mmol/L NaCl, 1 mmol/L NaH2PO4, 4 mmol/L Na2HPO4, 4 mmol/LKCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 5 mmol/L glucose [pH 7.4]).Transport measurements were performed in phosphate-bufferedringer at pH 7.4 and 37°C.
Glutarate Preincubation Experiments.
The preincubation scheme used in glutarate preload experimentswas the following: Cells were incubated in PBS containing 5mM glutarate (pH 7.4) for 50 min. After that, the cells wereincubated with the respective activators or inhibitors in thepresence of glutarate for 10 min. After that, the cells werewashed twice with ice-cold PBS and OA uptake was determinedafterward. Obtained trans-stimulation by glutarate preload wasnearly threefold. Trans-stimulation means uptake of a substratein exchange with a counter ion from the other (trans) side ofthe cell membrane. Thus, cis inhibition herein stands for acuteinhibition of substrate uptake by a compound located at thesame (cis) side of the cell membrane.
Tracer Flux Method.
The concentrations of the radiolabeled substrates applied tothe basolateral bath were as follows: 15 · 10-6 mol/L[14C]PAH and 55 · 10-9 mol/L [3H]mannitol. Mannitol wasused to correct secretion for paracellular fluxes and to determineextracellular water space. After 1 min in the transport buffer,the cells were washed twice with ice-cold PBS. Subsequently,the filters containing the cells were washed twice with ice-coldPBS and cut from the supports. Radioactivity of the cells wasmeasured using a liquid scintillation counter (Packard Instruments,Frankfurt, Germany). Counts of cells on filters were correctedfor nonspecific binding on filters by subtraction.
Fluorescein Uptake Method.
OA transport was also determined by measuring the uptake of10-6 M fluorescein after 1 min from the basolateral bath, accordingto a modified protocol (34); 10-6 M was used because of thebetter signal-noise ratio compared with 10 · 10-6 M fluorescein.After that, cells were washed six times with ice-cold PBS untilno fluorescein was detectable in the washing solution. Afterthat, the cells were lysed in 1 ml of 0.1% Triton-X100 in 20mM 3-(N-morpholino)propanedulfonic acid and fluorescence wascounted in a multiwell plate reader (Victor2, Wallac Instruments,Finland). Counts were corrected for extracellular binding andunspecific adhesion to the cells by subtraction of fluoresceincounts on cells at 4°C. Fluorescein counts were normalizedto protein content in the lysate measured by BCA protein assay(Pierce, Rockford, IL).
Compatibility of the Methods.
PAH uptake under control conditions was 29 pmol/mg per min (±5.37),and fluorescein uptake was 0.9 pmol/mg per min (±0.353).Data are presented as mean of all experiments (±SEM).The amount of [14C]PAH in the cells is 15-fold the amount offluorescein, which perfectly reflects the 15-fold higher concentrationin the basolateral bath. Thus, as [14C]PAH and fluorescein behavedidentically, the transport data were pooled and are all presentedas OA uptake in percentage of control. The method used for generationof the data is given in the respective figure legend. With respectto the action of PGE2, uptake of [14C]PAH or fluorescein alsobehaved identically. As for example in Figure 4A, PGE2 stimulationof OA uptake consists of PGE2 stimulation of [14C]PAH uptaketo 154% (±15.6; n = 5) of control and of PGE2 stimulationof fluorescein uptake to 195% (±62.5; n = 6) of control.Both statistically different from control but not from eachother.
Figure 4. Effect of inhibition of protein kinase A (PKA) by H89 on basolateral OA uptake in OK cells in the absence or presence of PGE2 or forskolin. Preincubation and uptake measurement was performed as mentioned in Materials and Methods. Cells were treated with PGE2 (A)/forskolin (B), MDL-12,330A alone, or PGE2 (A)/forskolin (B) in combination with MDL-12,330A for 10 min, respectively. After washing, uptake of OA was determined after 1 min. *P < 0.05 versus the respective control. (A) Effect of H89 (1 µM) on the PGE2 (100 nM) mediated stimulation of basolateral OA uptake. Substrate used was [14C]PAH or fluorescein l n = 10 to 11. (B) Effect of H89 (1 µM) on the forskolin (5 µM) mediated stimulation of basolateral OA uptake. Substrate used was [14C]PAH or fluorescein; n = 11 to 12.
Reverse Transcription–PCR
RNA from OK cells was extracted using Trizol reagent (Invitrogen,Carlsbad, CA). In brief, reverse transcription–PCR (RT-PCR)was performed according to Superscript One-Step RT-PCR systemprotocol (Invitrogen) with a final MgSO4 concentration of 1.45mM. cDNA was generated at 55°C for 15 min, then the sampleswere denatured at 94°C for 2 min. PCR amplification wasperformed in 35 cycles of 94°C for 15 s, then 55°C for30 s and 72°C for 30 s. For EP4, primers were 5'-GGAAGACTGTGCTCAGTA-3'(sense) and 5'-GAAGCAAATTCTTGCCTC-3' (antisense), covering bases1007 to 1246 of rat EP4 cDNA, 240 bp. For EP2, primers were5'-TTCGGAGCAAAAGAAGCC-3' (sense) and 5'-GAGCGCATTAGTCTCAGG-3'(antisense), covering bases 725 to 1025, 301 bp, both accordingto Jensen et al. (35).
PCR.
RT-PCR products were re-amplified using Platinum Taq polymerasekit (Invitrogen). MgSO4 concentration was 1.5 mM. PCR amplificationwas performed in 35 cycles of 94°C for 15 s, then 55°Cfor 30 s and 72°C for 30 s. EP4 primers were used as describedabove.
Generation of Sequence Data
PCR product of choice was eluated (Qiagen Gel Extraction Kit,Qiagen, Germany) and then cloned into the pCR3.1 vector usingInvitrogen TA cloning Kit. Sequencing was done by MWG Biotech(Munich, Germany). Homology search was done using BLAST. Homologywas quantified using ALIGNplus software.
Statistical Analyses
If not stated otherwise, data are presented as mean ±SEM. n is given in the text or in the figures. n is the numberof culture plates or filters used to perform the measurements.Statistical significance was determined by unpaired t test orANOVA as appropriate. Results were considered statisticallydifferent at P < 0.05. Significant difference is indicatedby asterisks.
Materials
[14C]PAH and [3H]mannitol were purchased from American RadiolabeledChemicals (St. Louis, MO). PD98059 and H89 were from AlexisCorp. (Läufelfingen, Switzerland). Butaprost and 11-deoxy-PGE1were from Cayman Chemical (Ann Arbor, MI). MDL-12,330A was fromCalbiochem (Merck, Darmstadt, Germany). If not stated otherwise,all other chemicals were from Sigma (St. Louis, MO).
PGE2 cis Inhibits Basolateral OA Uptake
It is well known that prostaglandins are substrates for theproximal tubular OA transport system and in particular for thebasolateral rate-limiting step mediated by OAT1 (31) or, asindicated only recently, OAT3 (15,32). To address the questionof how PGE2 stimulates OA uptake in OK cells, we investigatedwhether PGE2 acutely interacts with OA uptake, because if itdoes, then PGE2 may also act as a counter ion for OA uptake.As shown in Figure 2, PGE2 acutely inhibits basolateral OA uptakein OK cells. (For control uptake rates of PAH and fluorescein,see Transport Measurements and Compatibility of the Methods).According to its acutely inhibitory potency and the mentionedliterature, PGE2 may possibly serve as counter ion for OA uptake.Thus, a trans-stimulatory action of intracellularly generatedPGE2 increasing basolateral OA uptake rate cannot be ruled outup to this point.
Figure 2. Acute effect of PGE2 or forskolin on basolateral OA uptake in OK cells. Experiments were performed as described in Materials and Methods. Uptake of OA was measured after 1 min in the absence (control) or presence of 100 nM PGE2 or 5 µM forskolin in the basolateral bath solution containing the competing OA (here [14C]PAH); n = 6; *P < 0.05 versus control.
PGE2 Action Is Totally Abolished by Inhibition of Adenylate Cyclase
PGE2 stimulation of OA uptake cannot be mediated via EP1, asper the literature (22,23,36) and our own data (25), activationof PKC inhibits basolateral uptake of OA. Because in all possiblereceptor-signaling pathways remaining adenylate cyclase is involved,we investigated the effect of adenylate cyclase inhibition (byMDL-12,330A) on PGE2-mediated stimulation of basolateral OAuptake in OK cells. MDL-12,330A completely abolished the stimulatoryeffect of PGE2, whereas MDL-12,330A alone had no effect on basolateralOA uptake in OK cells (Figure 3). In addition, stimulation ofadenylate cyclase activity by forskolin (5 µM) led toan increase of OA uptake rate in OK cells (Figure 4B), whereasforskolin did not have a competitive effect on OA uptake (Figure 2).This is the first evidence that PGE2 does not act by a trans-stimulatorymechanism. In addition, as EP3 acts by adenylate cyclase inhibition,EP3 can be excluded as a possible receptor mediating PGE2 action.
Figure 3. Effect of inhibition of adenylate cyclase by MDL-12,330A (26.5 µM) on stimulation of basolateral OA uptake by PGE2 (100 nM) in OK cells. Cells were treated with PGE2, MDL-12,330A alone, or PGE2 in combination with MDL-12,330A for 10 min, respectively. Then, basolateral uptake of OA (here [14C]PAH) after 1 min was determined; n = 7 to 10; *P < 0.05 versus control.
Effect of PGE2 or Forskolin Is Abolished by Inhibition of PKA
Increased activity of adenylate cyclase leads to generationof cAMP, which then activates PKA. Thus, we investigated whetherthe increase of OA uptake induced by PGE2 or forskolin is affectedby inhibition of PKA activity (1 µM H89). Inhibition ofPKA totally abolished the effect of both PGE2 (Figure 4A) andforskolin (Figure 4B) on OA transport, whereas H89 alone hadno effect. This is additional evidence that PGE2 action is notdue to trans-stimulation.
PGE2 or Forskolin also Stimulates OA Uptake in Glutarate-Preloaded Cells
To gain more information on whether the effects of PGE2 or forskolinare partly trans-stimulatory, we investigated their effect inglutarate-preloaded cells. OK cells were preloaded with glutarate(5 mM; 50 min), a nonmetabolizable analogue of -ketoglutaratethat drives OA uptake under physiologic conditions. As shownin Figure 5, preloading the OK cells with glutarate leads toa nearly threefold increase of OA uptake rate. Under this condition,100 nM PGE2 (Figure 5A) or 5 µM forskolin (Figure 5B)still stimulated OA uptake. The latter stimulation was stillinhibitable by inhibition of PKA in both cases (Figure 5). Thisis additional important evidence that PGE2 stimulates OA uptakevia a mechanism different from trans-stimulation.
Figure 5. Effect of glutarate preload on basolateral OA uptake in OK cells and its stimulation by PGE2 or forskolin. Incubation was performed as mentioned in Materials and Methods. In brief, cells were incubated with 5 mM glutarate for 50 min. Then, cells were treated with PGE2/forskolin, H89 alone, or PGE2/forskolin in combination with H89 for 10 min in the presence of glutarate. After intense washing, uptake of OA was determined after 1 min. Data are presented as percentage of the unstimulated (not glutarate-preincubated) control. Thus, glutarate control indicates cells solely preincubated with glutarate. Substrates used were [14C]PAH or fluorescein. *P < 0.05 versus the respective control. (A) Effect of H89 (1 µM) on the PGE2 (100 nM) mediated stimulation of basolateral OA uptake in OK cells preloaded with glutarate (5 mM; 50 min); n = 6 to 9. (B) Effect of H89 (1 µM) on the forskolin (5 µM) mediated stimulation of basolateral OA uptake in OK cells preloaded with glutarate (5 mM; 50 min); n = 6 to 9.
MAPK Are Not Involved in PGE2 or Forskolin Action
In OK cells, it is described that activation of the MAPK ERKis dependent on the cAMP level of the cells after 2 adrenergicstimulation (37). To investigate whether MAPK are involved indownstream signaling of PGE2 or whether feed back loops arepresent, we investigated the effect of inhibition of MEK by5 µM PD98059. As indicated in Figure 6A, neither was thestimulation of OA uptake by PGE2 affected by PD98059 nor did5 µM PD98059 alone have any effect. The same was truefor forskolin (Figure 6B). Thus, ERK1/2 activation is not involvedin PGE2-mediated stimulation of basolateral OA uptake in OKcells. This is in good agreement with our previous data (26)on signaling hierarchy, showing ERK upstream of prostaglandingeneration.
Figure 6. Effect of inhibition of ERK1/2 by PD98059 on basolateral OA uptake in OK cells in the absence or presence of PGE2 or forskolin. Incubation was performed as mentioned in Materials and Methods. In brief, cells were treated with PGE2 (A)/forskolin (B), PD98059 alone, or PGE2 (A)/forskolin (B) in combination with PD98059 for 10 min. OA used was [14C]PAH. *P < 0.05 versus the respective control; n = 6. (A) Effect of PD98059 (5 µM) on the PGE2 (100 nM) mediated stimulation of basolateral OA uptake. (B) Effect of PD98059 (5 µM) on the forskolin (5 µM) mediated stimulation of basolateral OA uptake.
Prostaglandins Act via EP2 or EP4
Following what was mentioned before, PGE2 should act by bindingto prostanoid receptor subtypes EP2 or EP4. Thus, we testedwhether the EP2,3,4 selective prostanoid 11-deoxy PGE1 stimulatesbasolateral OA uptake in OK cells. As shown in Figure 7A, 11-deoxyPGE1 (10 min) dose-dependently stimulates OA uptake. In addition,11-deoxy PGE1 did not show any acute, cis-inhibiting effecton OA uptake (data not shown). Taken together, this is additionalpharmacologic evidence that PGE2 acts via the EP2 or EP4 receptor.
Figure 7. Effect of selective EP receptor agonists on basolateral OA uptake in OK cells. OK cells were incubated with various concentrations of the respective agonist for 10 min. After washing, basolateral uptake of OA was determined after 1 min. OA used was fluorescein. *P < 0.05 versus the respective control. (A) Effect of increasing concentrations of 11-deoxy PGE1 on basolateral OA uptake; n = 8 to 17. (B) Effect of increasing concentrations of butaprost on basolateral OA uptake; n = 6 to 8.
Prostaglandins Act via a Homologue of EP4
To gain more detailed information about the nature of PGE2 action,we investigated whether the EP2 selective prostanoid butaprostaffects basolateral OA uptake in OK cells. Reasonable concentrationsof butaprost (Km = 100 nM) have no effect on OA transport rate(Figure 7B). Butaprost also has no competitive effect on OAuptake (data not shown). As no data on the presence or absenceof prostanoid receptors in OK cells are available, we determinedwhether an EP2 or EP4 analogue is detectable in OK cells. NoEP2 signal was detectable, whereas RT-PCR against EP4 (as describedin Materials and Methods) led to a fragment of approximately240 bp (Figure 8A). Sequencing led to a fragment of 239 bp inlength, which hit rat EP4 prostanoid receptor in BLAST search(Figure 8B) as well as the human type (see Discussion). Localhomology search using ALIGNplus software indicated a 64% homologyof the cloned fragment to the sequence segment predicted bythe primers used in PCR (Figure 8, B and C). Thus, most likely,an EP4 receptor homologue to which PGE2 can bind is presentin OK cells.
Figure 8. Detection of a rat EP4 receptor homologue in OK cells. (A) Reverse transcription–PCR (RT-PCR) product from OK cell mRNA. Primers against EP4 and conditions were as described in Materials and Methods. M, marker lane; EP4, lane with RT-PCR product. (B) Graph showing the location and grade of homology of the OK cell RT-PCR product to the rat EP4 receptor. (C) Alignment of the OK cell 239 bp RT-PCR product to the local homology region of rat EP4 receptor DNA sequence. EP4-1B1.TXT, RT-PCR product from OK cell mRNA; RATEP41:TXT, rat EP4 receptor DNA sequence.
EGF-Induced Stimulation of OA Uptake Is Mediated by Adenylate Cyclase and PKA
Finally, we investigated whether the stimulatory action of EGFon proximal tubular OA uptake described before (25,26) includesthe PGE2 mechanism presented here. Therefore, we investigatedthe effect of inhibition of PKA (by H89; Figure 9A) or adenylatecyclase (by MDL-12,330A; Figure 9B) on EGF-induced stimulationof basolateral OA uptake in OK cells. Both inhibition of PKAand adenylate cyclase completely abolished EGF-induced stimulationof OA uptake. Thus, the stimulatory mechanism of EGF on OA uptakeincludes the PGE2-induced mechanism presented herein.
Figure 9. Effect of inhibition of PKA (by H89) or adenylate cyclase (by MDL-12,330A) on the EGF-mediated stimulation of OA uptake in OK cells. EGF, H89, and MDL-12,30A alone or in the respective combination were applied for 10 min. After washing, basolateral uptake of OA was determined for 1 min. OA used was fluorescein. *P < 0.05 versus the respective control; n = 6 to 10. (A) Effect of H89 on EGF-mediated stimulation of basolateral OA uptake. (B) Effect of MDL-12,330A on EGF-mediated stimulation of basolateral OA uptake.
As already mentioned in the introduction, only little is knownabout the regulation of basolateral uptake of OA. In previousstudies, we reported that EGF stimulates basolateral OA uptakein OK cells via MEK and ERK1/2 (25), which then activates phospholipaseA2 (PLA2), leading to arachidonic acid generation. COX1 thentransforms arachidonic acid into PGE2, which dose-dependentlyactivates basolateral OA uptake (26). For this stimulation tooccur, two mechanisms are possible. First, PGE2 could act asa counter substrate and thus stimulate the uptake of OA. Second,PGE2 could act via coupling to specific receptors present atthe membrane of proximal tubular OK cells. In the following,both possibilities are discussed.
Literature (19,31,32) indicates that PGE2 is a substrate forthe basolateral OA uptake exchanger OAT1. PGE2 is also shownto be a substrate for the human type OAT3 (4). Our own data(Figure 2) show that PGE2 acutely interacts with OA uptake.Thus, the possibility emerges that PGE2 may cis-inhibit basolateraluptake of OA competitively (Figure 2) and may trans-stimulatebasolateral OA uptake when present inside the proximal tubularcells (for definition of the cis- and trans-nomenclature, seeMaterials and Methods). In addition, cAMP generated when PGE2binds to EP2 or EP4 receptors is a substrate for basolateralorganic uptake mechanisms (8,19). Thus, it may cis-inhibit andtrans-stimulate with respect to basolateral OA uptake. Therefore,we decided to investigate the action of PGE2 under circumstanceswhen trans-stimulation by glutarate occurs. Preincubation ofOK cells with 5 mM glutarate for 50 min is shown to increaseOA uptake approximately threefold (Figure 5). This trans-stimulatorymechanism is well described for OAT1 (7,8,19,38) and also forOAT3 (15). After preincubation with glutarate, PGE2 still showsstimulatory action. In detail, after preincubation, relativestimulation of basolateral OA uptake by the same total amountof PGE2 (100 nM) is approximately 1.7-fold compared with therespective control (Figure 5A; glutarate control). In cellsnot preloaded with glutarate (Figure 4A; control set as 100%),a 1.8-fold increase compared with the respective control occurred.Preincubation of OK cells with glutarate leads to an increaseof counter ions for basolateral OA uptake. Thus, if acting ina trans-stimulatory manner, the same amount of PGE2 should exerta decreased relative stimulatory action in cells preloaded withglutarate. As this is not the case, it is evident that PGE2action presented herein is not due to trans-stimulation. Inaddition, if stimulation by PGE2 was due to trans-stimulation,then inhibition of adenylate cyclase should not have any effecton its action. Moreover, PGE2 action was mimicked by adenylatecyclase activator forskolin in both control and glutarate-preloadedOK cells. Forskolin is not a potential counter ion for the basolateralOA exchanger as no acute inhibitory interaction with OA uptakewas observed (Figure 2). The PKA inhibitor H89 completely abolishedPGE2 action in both control and glutarate-preloaded OK cells,which is evidence that cAMP does not act as counter ion withrespect to stimulation of basolateral OA uptake. In addition,forskolin-induced stimulation of OA uptake persists after glutaratepreload as compared with the control cells (in parallel to whatwas mentioned before for PGE2). Moreover, forskolin action onOA uptake is completely abolished by inhibition of PKA (in controland glutarate-preloaded OK cells), which in addition contradictsa trans-stimulatory mechanism. Taken together, these data excludethe possibility of trans-stimulation as a putative mechanismof the dose-dependent PGE2 action on initial basolateral OAuptake in OK cells described by Sauvant et al. (26).
Thus, PGE2 action should be mediated by activation of one ofits receptors. Four isoforms of the PGE2 receptors, named EP1to EP4, are known, and all of them are present in the kidneyand act via G-proteins (35,39). EP1 is known to act via Gq afteractivation of PKC. As already mentioned in the introduction,it is well known from literature and from our own experimentsthat activation of PKC inhibits basolateral OA uptake. Thus,in our case, EP1 is not a possible receptor for mediation ofPGE2 action. The remaining possibilities include EP2 and EP4,both activating adenylate cyclase and PKA, or EP3, which inhibitsadenylate cyclase and PKA. If PGE2 would stimulate OA uptakevia EP3, then inhibition of adenylate cyclase or PKA shouldstimulate uptake as activation of EP3 leads to inhibition ofadenylate cyclase and PKA. However, as indicated in Figures 3 and 4, both maneuvers mentioned have no effect on OA uptakein OK cells. In addition, if PGE2 would stimulate OA uptakein OK cells via the EP3 receptor, then inhibition of both adenylatecyclase and PKA in the presence of PGE2 should lead to increasedstimulation as compared with PGE2 action alone. As this alsois not the case, it seems highly unlikely that PGE2 is actingvia the EP3 receptor subtype. This is confirmed by the factthat forskolin stimulates OA uptake and that this stimulationis again prevented by inhibition of PKA. Thus, stimulation ofOA uptake by PGE2 cannot be mediated by a mechanism inhibitingadenylate cyclase or PKA, as EP3 receptor stimulation does.
Thus, the stimulatory action of PGE2 should be mediated by interactionwith the EP2 or EP4 receptor subtype. This hypothesis was confirmedusing 11-deoxy PGE1, an EP2,3,4 selective agonist (Km approximately30 to 50 nM for EP2 or EP4 (40)). Figure 7 shows the stimulatoryaction of 11-deoxy PGE1 on basolateral OA uptake. In addition,no acute inhibitory interaction with OA uptake was observedfor 11-deoxy PGE1 at a concentration being at least twice ashigh as affinity constant (40). Thus, 11-deoxy PGE1 is not apossible substrate or a counter ion with respect to basolateraluptake of OA. 11-Deoxy PGE1 has also been described to bindto EP3 with high affinity (Km approximately 1.5 nM (40)), butas a result of the observed stimulation of OA uptake by 11-deoxyPGE1 and the above-mentioned data, excluding EP3 as a possiblereceptor for mediating stimulation of OA uptake, this possibilitycan be ruled out. In addition, reasonable amounts of the EP2selective agonist butaprost (Km approximately 100 nM; (40))do not affect basolateral organic uptake rate, neither after10 min nor acutely. The EP selective agonists used were shownto act not only in rodents but also in cells from pigs (41)and humans (15). Thus, their pharmacologic profile seems notto be strictly species dependent. Thus, all pharmacologic datapresented give evidence that the receptor-mediating stimulationof basolateral OA uptake by PGE2 is of the EP4 type.
As no molecular evidence for the presence of EP receptors inOK cells was available, we decided to determine whether homologuesto the known rat receptor subtypes EP2 or EP4 are expressed.As mentioned in Results, no mRNA for EP2 could be detected inOK cells with the methods used herein. Certainly, this is notproof for the absence of EP2 in OK cells. However, these dataare in good agreement with the fact that butaprost has no effecton OA uptake. However, using EP4 selective primers and RT-PCR,we were able to clone a 239-bp fragment showing 64% homologyto the respective part of the rat EP4 receptor DNA sequence.Thus, an mRNA homologue to EP4 is expressed in the proximaltubular OK cell line. Moreover, the cloned EP4 fragment fromOK shows not only 64% homology to the respective part of theEP4 receptor DNA sequence from rat but also a 67% homologueto respective human sequence. Comparison of the EP4 receptorsequences from rat and human leads to a homology of 65%. Thus,the OK cell fragment cloned is as homologous to rat or humanas the EP4 receptors from rat and human are compared with eachother. Together with the pharmacologic data of butaprost and11-deoxy PGE1 presented above, this is clear evidence that prostanoidsstimulate basolateral OA uptake in OK cells by a mechanism activatingadenylate cyclase and PKA. This is in good agreement with datashowing an increase in cAMP in human proximal tubular cells(42) after incubation with PGE2. The receptor mediating thisregulatory cascade is most likely a homologue of the EP4 receptorsdescribed in rats or humans.
As already mentioned, EGF-mediated stimulation of basolateralOA transport in OK cells is due to a signaling pathway leadingto generation of PGE2 (26). Herein PGE2 was shown to stimulateOA uptake by activation of PKA and adenylate cyclase. In addition,EGF stimulation of OA uptake was abolished by inhibition ofadenylate cyclase or PKA. Thus, PGE2 generation links EGF actionand PKA activation. Moreover, this is again evidence contradictinga trans-stimulatory mechanism of PGE2, because in this case,EGF action should not be abolished by the above-mentioned inhibitorymechanisms.
The observed stimulatory effects of PGE2 on initial basolateraluptake of OA presented before (26) can be explained by usingthe following model (Figure 10): PGE2 is generated in responseto EGF (via a signaling pathway shown by Sauvant et al. (26))in the cells or externally, then most likely binds to a EP4receptor leading to G-protein–mediated activation of adenylatecyclase. cAMP then activates PKA, which stimulates basolateralOA uptake by an as-yet-unknown mechanism. Meanwhile, PKA phosphorylationsites were shown to exist on human OAT1 but up to now are notindicated in human or rat OAT3 (32). However, this does notexclude the possibility that OAT3 is stimulated by PKA as thisaction may also be mediated via the phosphorylation of auxiliaryproteins interacting with the respective transport protein.In addition, You et al. (24) showed that PKC inhibits murineOAT without direct phosphorylation of the transport proteinitself. Thus, it has to be further investigated whether a stimulationof OAT1 and/or OAT3 is responsible for increased OA uptake inproximal tubular cells after EGF or PGE2.
Figure 10. Summary showing the mechanism of prostaglandin action on basolateral OA uptake in OK cells in the short time range. PGE2 binds to the EP4 receptor homologue, which leads to an increase in adenylate cyclase activity. Thus, the generation of cAMP is enhanced, leading to an increase in PKA activity. PKA then stimulates the basolateral OA uptake by an as-yet-unknown mechanism. For reasons mentioned in Discussion, OAT1 and OAT3 are the most likely candidates for the observed stimulation. EGF stimulation of basolateral OA uptake described before is shown to be mediated via the mentioned mechanism.
PGE2 plays a crucial role in inflammation (43). EP4 receptorshave been detected in the proximal tubule (35,39). In addition,PGE2 is considered to be the major renal cyclooxygenase metaboliteof arachidonic acid (27), which is known to be produced in therenal cortex (44) and in particular in proximal tubular cells(45). In inflammatory disease of the kidney, PGE2 and its receptorsare shown to be upregulated (28). In proximal tubular cellsfrom pigs and humans, PGE2 is generated by reactive oxygen species(42,46) induced by stress and may directly contribute to renallesions and loss of kidney function. As basolateral uptake isconsidered to be the rate-limiting step of OA secretion, weexpect that PGE2 induces stimulation of OA secretion into theproximal tubular lumen. As PGE2 is an OA itself, this mechanismmay reduce the concentration of PGE2 in the kidney interstitium,which contradicts its inflammatory potency. We hypothesize thatthis may represent a mechanism to limit PGE2-induced inflammatoryevents in the kidney cortex interstitium. Therefore, we furtherhypothesize that inhibition of OA secretion during kidney inflammationmay contribute to an increase in severity of inflammation. Thiswill have to be investigated in further experiments.
Acknowledgments
This work was supported by the Deutsche ForschungsgemeinschaftDFG grant Ge 905/3-4.
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Received for publication November 5, 2003.
Accepted for publication August 27, 2003.
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Top
Abstract
Introduction
-ketoglutarate, which is then exchanged for OA (2,3,5,6). The basolateral exchanger for OA and dicarboxylates was cloned by three independent groups (7–9) in 1997 and named OAT1. The homologous protein was cloned from human kidney and was called hOAT1 (10,11) or hPAHT (12). The genomic DNA from human hOAT1 is organized in 10 exons, and up to now, four isoforms have been described (13). Furthermore, it was shown that OAT1 represents characteristics of the basolateral, polyspecific transporter for OA (14), which has been functionally described for a substantial amount of time (6). Meanwhile, there is evidence that the basolaterally located OAT3 from rat also acts as an exchanger for dicarboxylates and OA as OAT1 does (15). 
, both maneuvers mentioned have no effect on OA uptake in OK cells. In addition, if PGE2 would stimulate OA uptake in OK cells via the EP3 receptor, then inhibition of both adenylate cyclase and PKA in the presence of PGE2 should lead to increased stimulation as compared with PGE2 action alone. As this also is not the case, it seems highly unlikely that PGE2 is acting via the EP3 receptor subtype. This is confirmed by the fact that forskolin stimulates OA uptake and that this stimulation is again prevented by inhibition of PKA. Thus, stimulation of OA uptake by PGE2 cannot be mediated by a mechanism inhibiting adenylate cyclase or PKA, as EP3 receptor stimulation does. 
1.TXT, RT-PCR product from OK cell mRNA; RATEP4
