A Transporter of Ibuprofen is Upregulated in MDCK ... - ACS Publications

Jul 9, 2016 - Universitetsparken 2, DK-2100 Copenhagen, Denmark. §. Pharmaceutical Science and CMC Biologics, H. Lundbeck A/S, DK-2500 Valby, ...
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A Transporter of Ibuprofen is Upregulated in MDCK I Cells under Hyperosmotic Culture Conditions Carsten Uhd Nielsen,*,† Rune N. Rasmussen,† Junying Mo,‡ Benafsha Noori,‡ Candela Lagunas,‡ René Holm,§,∥ and Martha K. Nøhr‡ †

Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark Drug Transporters in ADME, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark § Pharmaceutical Science and CMC Biologics, H. Lundbeck A/S, DK-2500 Valby, Denmark ‡

S Supporting Information *

ABSTRACT: Ibuprofen is a widely used drug. It has been identified as an inhibitor of several transporters, but it is not clear if ibuprofen is a substrate of any transporter itself. In the present work, we have characterized a transporter of ibuprofen, which is upregulated by hyperosmotic culture conditions in Madin−Darby canine kidney I (MDCK I) renal cells. [3H]-Ibuprofen uptake rate was measured in MDCK I cell cultured under normal (300 mOsm) and hyperosmotic (500 mOsm) conditions. Hyperosmotic conditions were obtained by supplementing urea, NaCl, mannitol, or raffinose to culture medium. The effect of increased osmolarity was investigated for different incubation times. [3H]-Ibuprofen uptake in MDCK I cells was upregulated by hyperosmotic culture condition, and was saturable with a Km value of 0.37 ± 0.08 μM and a Vmax of 233.1 ± 17.2 pmol· cm−2· min−1. Racemic [3H]-ibuprofen uptake could be inhibited by (R)-(−)- and (S)-(+)-ibuprofen with IC50 values of 19 μM (Log IC50 1.39 ± 0.34) and 0.47 μM (Log IC50 −0.36 ± 0.41), respectively. Furthermore, the [3H]-ibuprofen uptake rate was increased by decreased extracellular pH but not dependent on Na+ or Cl− ions. The mRNA of Mct1, -2, -4, and -6 as well as Oat1 and -3 were not upregulated by hyperosmolarity. Our findings present strong evidence for the presence of a yet unknown ibuprofen transporter in MDCK I cells. The transporter was upregulated under hyperosmotic culture conditions, and the present study is therefore a starting point for identification of the molecular correlate and potential impact on ibuprofen disposition. KEYWORDS: ibuprofen, MDCK 1 cells, osmolarity, transporter, upregulation

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INTRODUCTION Ibuprofen is a nonsteroidal anti-inflammatory drug substance used to treat pain, fever, and inflammation. It is on the WHO list of essential medicines, a list made to suggest the basic medicines required in a health care system.1 Ibuprofen (as the racemate, i.e. equal amounts of (R)-(−) and (S)-(+)-ibuprofen) is administered orally in doses of 200−600 mg 1−3 times daily. The absorption is almost complete and controlled by the pH gradient from the mucus microclimate to the cell facilitating absorption of the uncharged ibuprofen species.2,3 The absorption results in maximal plasma concentrations of ibuprofen ranging between 15 and 50 μg/mL, equal to 70−240 μM (extracted from ref 4). The maximum plasma concentration is reached within 1−2 h after administration, and 70−90% of the ibuprofen dose administered is recovered as ibuprofen or its metabolites in the urine over 24 h after administration.4,5 The amount of free ibuprofen excreted in the urine after oral administration of ibuprofen is generally low, 98% binding,6 which has been shown to influence the disposition of ibuprofen to the brain.7 Ibuprofen has been identified as an inhibitor of substrate transporters via a number of solute carriers (SLCs) such as the proton-coupled di/tripeptide transporter hPEPT1 (SLC15A1),3 the sodium-coupled monocarboxylate transporter 1 (SMCT1, SLC5A8), 8 the organic anion transporter 1 OAT1 (SLC22A6),9,10 rOat2,11 and hOAT1−4,12 and the organic cation transporters OCT1 and OCT2.12 Moreover, ibuprofen has been shown to inhibit lactate transport in CHO and BoWe Received: April 13, 2016 Revised: May 26, 2016 Accepted: July 9, 2016

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DOI: 10.1021/acs.molpharmaceut.6b00330 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics cells.13,14 Lactate is a substrate of monocarboxylate transporters such as, e.g., MCT1, MCT2, and MCT4.15 The uptake of lactate in CHO cells has been reported to be inhibited to a higher extend by (R)-(−)-ibuprofen than by (S)-(+)-ibuprofen at identical concentrations of the enantiomers.13 Ibuprofen also inhibited the uptake via ATP-binding cassette transporters (ABCs) of methotrexate via MRP2 (ABCC2) and MRP4 (ABCC4) in membrane vesicles from MDCK II/MRP2 and HEK293/MRP4 cells, respectively.16 Despite the extensive inhibiting effect of ibuprofen on a number of transporters, only two indications have to the best of our knowledge suggested actual transportermediated uptake of ibuprofen. It has been shown that ibuprofen transport across the rat blood−brain barrier was saturable at increasing ibuprofen concentrations, why a carrier-mediated uptake component was suggested to be involved in brain uptake.7 Also, the uptake of 0.5 μM ibuprofen in hOAT1 and hOAT3 transfected S2 cells was 1.4 and 1.7 times higher than in the corresponding mock S2 cells, which also suggested a transporter mediated uptake.12 Collectively, ibuprofen interacts with a number of transporters as an inhibitor, whereas no clear identification or characterization of a transporter that mediates the cellular transport of ibuprofen in the kidney has been performed. The potential impact of transporters on ibuprofen distribution and renal excretion therefore remains unknown. During our investigations with effects of hyperosmolarity, we discovered that ibuprofen uptake was increased in hyperosmotic treated MDCK I cells, which is a commonly used renal cell line strain isolated from the parental canine kidney cell line.17 The aim of the present study was therefore to investigate if this increased uptake was due to transporter-mediated uptake and to characterize the transporter kinetics in MDCK I cells. Here we have identified that a so far functionally uncharacterized ibuprofen transporter is upregulated under hyperosmotic culture conditions in MDCK 1 cells.

from Corning Life Science (Wilkes Barre, PA, USA). Water was used from a Milli-Q water purification system. Foetal bovine serum (FBS) was from Thermo Fisher Scientific (Waltham, MA). (S)-(+)-Ibuprofen and (R)-(−)-ibuprofen were kind gifts from Steen H. Hansen (University of Copenhagen). Alexa 488− phalloidin and propidium iodide were from Molecular Probes (Leiden, The Netherlands). Cell Cultivation. MDCK I cells obtained from European Collection of Authenticated Cell Cultures (ECACC, Netherlands) were cultured using DMEM/F-12 containing 1% penicillin−streptomycin, 2 mM L-glutamine, and 10% FBS (later termed “growth medium”) in an atmosphere of 5% CO2− 95% O2 at 37 °C. MDCK I cells were used at passages 3−8. The cells were cultured in noncoated 24-well plates at a density of 5.7 × 104 cells·mL−1 (total of 1.14 × 105 cells per well) for a total of 144 h. The last 24 h (unless otherwise stated) of the culturing period, the cells were incubated with growth medium (having an osmolarity of 300 mOsm) supplemented with mannitol, urea, raffinose, or NaCl, respectively, to a total final osmolality of 500 mOsm in the medium. Control cells were cultured in normal growth medium (300 mOsm). Cellular Transport Experiments. Buffer Solutions. Uptake studies were performed in HBSS (in mM: CaCl2, 1.26; MgCl2, 0.49; MgSO4, 0.41; KCl, 5.33; KH2PO4, 0.44; NaCl, 138; Na2HPO4, 0.34; D-glucose, 5.56; NaHCO3, 4.5). The HBSS solutions were buffered with 10 mM HEPES or MES and pH adjusted to 7.4 or 6.0, respectively, with NaOH or HCl. Sodium free HBSS was prepared by substituting sodium with choline (in mM: CaCl2, 1.3; MgCl2, 0.5; MgSO4, 0.4; KCl, 5.4; KH2PO4, 0.4; C5H14ClNO, 137.5; K2HPO4, 0.34; D-glucose, 5.56) and buffered with 10 mM of HEPES to pH 7.4. Chloride free HBSS was prepared by substituting chloride with gluconate (in mM: CaC12H22O14, 1.3; MgC12H22O14, 0.5; MgSO4, 0.4; KC6H11O7, 5.4; KH2PO4, 0.4; NaC6H11O7, 137.5; Na2HPO4, 0.34; D-glucose, 5.56) and buffered with 10 mM of HEPES to pH 7.4. The cells were equilibrated in prewarmed experimental buffers at 37 °C for 15 min before the studies. Uptake of Ibuprofen in MDCK I Cells. The cellular uptake of [3H]-ibuprofen (1 μCi·mL−1, 200 nM, 5 Ci·mmol−1) in MDCK I cells cultured under hyperosmotic (mannitol; 500 mOsm) and control (300 mOsm) conditions was measured. The cells were incubated for 10 min at 37 °C with [3H]-ibuprofen in HBSS buffer, pH 7.4. Additionally, the uptake of [3H]-ibuprofen was measured in the presence of 1 mM unlabeled ibuprofen. After incubation, the uptake medium was removed from the cells and the cells were washed three times with ice-cold HBSS buffer. The cells were detached from the wells by addition of 200 μL 0.1% Triton-X 100 in H2O. Additionally, the uptake of [3H]ibuprofen (0.25 μCi·mL−1, 50 nM, 5 Ci·mmol−1) in MDCK I cells cultured under hyperosmotic (mannitol, urea, NaCl, or raffinose (500 mOsm)) and control (300 mOsm) conditions were measured. The cells were incubated for 5 min at 37 °C with [3H]-ibuprofen in HBSS buffer, pH 7.4. Additionally, the uptake of [3H]-ibuprofen was measured in the presence of 100 or 250 μM unlabeled ibuprofen. The cellular uptake of [3H]-ibuprofen in MDCK I cells cultured under hyperosmotic (mannitol or raffinose) conditions (500 mOsm) was further investigated in the absence of sodium or chloride in the uptake buffer and in the presence of various substrates for other transporters (see figure legends for details). For mannitol induced hyperosmolality, the [3H]-ibuprofen concentration was 0.5 μCi·mL−1 (100 nM, 5 Ci·mmol−1) and the uptake time 10 min. For raffinose induced hyperosmolality,

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MATERIAL AND METHODS Chemicals and Reagents. Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12, ibuprofen, mannitol, 2-(Nmorpholino)ethanesulfonic acid (MES), Bradford reagents, nigericin sodium salt, D-(+)-raffinose pentahydrate, mono potassium phosphate, magnesium gluconate, Triton-X100, magnesium sulfate heptahydrate, magnesium chloride hexahydrate, calcium chloride dihydrate, choline chloride, acetylsalicylic acid, piroxicam, urea, probenecid, 5-amino salicylic acid (5-ASA), folic acid, L-glutamic acid, quercetin dihydrate, D,L-lactic acid, racemic ibuprofen, and penicillin−streptomycin were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Potassium chloride, potassium dihydrogen phosphate, D-glucose, potassium gluconate, and sodium gluconate were obtained from Merck KGaA (Darmstadt, Germany). Bovine serum albumin (BSA) was from Biotech Line (Slagelse, Denmark). Fluvastatin was purchased from AH Diagnostics (Aarhus, Denmark). 4-(2Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was obtained from AppliChem GmbH (Darmstadt, Germany). Hank’s Buffered Saline Solution (HBSS) (10×) and sodium bicarbonate (7.5%) were obtained from Gibco, Invitrogen (Pairsley, UK). Calcium gluconate and sodium chloride were purchased from VWR−Bie & Berntsen (Herlev, Denmark). RS[Ring-3H] ibuprofen ([3H]-ibuprofen; with two different batches with specific activities of 5 or 20 Ci·mmol−1) was from American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA). Ultima Gold scintillation liquid was purchased from PerkinElmer (Boston, MA, USA). Cell culture plastic ware was obtained B

DOI: 10.1021/acs.molpharmaceut.6b00330 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics the [3H]-ibuprofen concentration was 0.5 μCi·mL−1 (25 nM, 20 Ci·mmol−1) and the uptake time 5 min. The proton dependency of the cellular uptake of [3H]ibuprofen (0.5 μCi·mL−1, 100 nM, 5 Ci·mmol−1) in MDCK I cells cultured under hyperosmotic (mannitol) and normal condition cells was determined. The uptake was measured at pH 6.0 and 7.4, and furthermore in the presence of 250 μM unlabeled ibuprofen. The uptake time was 10 min. The proton dependency of the cellular uptake of [3H]-ibuprofen was also investigated in raffinose-induced hyperosmolality in MDCK I cells. The [3H]-ibuprofen concentration was 0.5 μCi·mL−1 (25 nM, 20 Ci·mmol−1). The uptake was measured at both pH 6.0 and 7.4, and, additionally, for both conditions the uptake was measured in the presence of 100 μM unlabeled ibuprofen, 20 μM nigericin, or 100 μM ibuprofen and 20 μM nigericin, respectively. When the cells were treated with nigericin, an addition of 150 mM KCl was supplemented to the uptake buffer. The uptake time was 10 min. The uptake of [3H]-ibuprofen (0.6 μCi·mL−1, 30 nM, 20 Ci· mmol−1) in MDCK I cells cultured under hyperosmotic conditions (raffinose) was measured in the presence of unlabeled 0.5 μM (R)-(−)-ibuprofen or 0.5 μM (S)-(+)-ibuprofen, respectively. Furthermore, the concentration dependent inhibition of the [3H]-ibuprofen (0.6 μCi·mL−1, 30 nM, 20 Ci· mmol−1) was determined in the presence of unlabeled (R)(−)-ibuprofen (0.5−100 μM) or (S)-(+)-ibuprofen (0.001−2.5 μM). The uptake time was 5 min. The concentration dependent ibuprofen uptake in MDCK I cells cultured under hyperosmotic (raffinose or mannitol) and normal conditions was determined in the presence of [3H]ibuprofen, and unlabeled ibuprofen was used to adjust the total extracellular concentration. The concentration of [3H]-ibuprofen used for raffinose-induced hyperosmolality and isoosmolality (300 mOsm) cellular uptake was 0.125 (6.25, 20 Ci· mmol−1 nM) or 0.25 μCi·mL−1 (12.5 nM, 20 Ci·mmol−1), respectively and the uptake time 5 min, and for mannitol-induced hyperosmolality 0.125−1 μCi·mL−1 (25−200 μM, 5 Ci·mmol−1) [3H]-ibuprofen and the uptake time 10 min. The effect of the culture time under hyperosmotic condition on the cellular uptake of [3H]-ibuprofen in MDCK I cells was determined. The cells were first cultured in normal medium and afterward treated with growth medium supplemented with raffinose (500 mOsm). The exposure time of the cells to the hyperosmotic medium was 0−48 h, with a total culture time of 144 h. After the exposure, the uptake of [3H]-ibuprofen (0.6 μCi· mL−1, 30 nM, 20 Ci·mmol−1) was determined, with an uptake time of 5 min. Additionally, the cellular uptake of [3H]-ibuprofen was also determined when the MDCK I cells were first cultured under hyperosmotic (raffinose, 500 mOsm) condition for 24 h and then afterward cultured for 2 to 24 h under isotonic condition (300 mOsm) before the uptake of [3H]-ibuprofen was determine. The cellular uptake rate of [3H]-ibuprofen (0.6 μCi·mL−1, 30 nM, 20 Ci·mmol−1) was determined in MDCK I cells cultured under hyperosmotic (500 mOsm; raffinose) or iso-osmotic (300 mOsm) conditions with the supplement of either 50 μM ibuprofen, 50 μM acetylsalicylic acid, or 10 μM piroxicam, respectively, for the last 24 h before the uptake experiment. Real-Time Polymerase Chain Reaction. Real-time PCR was performed on mRNA isolated from MDCK I cells cultured for the final 24 h of culture under hyperosmotic (urea, NaCl, or raffinose, 500 mOsm) and iso-osmotic conditions (300 mOsm). Total mRNA was isolated with Nucleospin RNA/Protein

(Machenerey-Nagel GmbH Co., Düren, Germany)), cDNA was obtained using TaqMan reverse transcription reagents (Applied Biosystem, Foster City, CA, USA), and SsoFast EvaGreen super mix (2×) (Biorad laboratories, Denmark) was used for real time PCR. The experiments were performed with two technical replicates for each 4−5 biological replicates (4−5 independent cell passages). Primers for the selected canine transporters are shown in the Supporting Information. Hypoxanthine-guanine phosphoribosyltransferase (Hprt) was used as a housekeeping gene. The primers were obtained from Eurofins Genomics (Ebersberg, Germany). Morphological Studies of MDCK I Cells Cultured under Hyperosmotic Conditions. The morphology of MDCK I cells cultured under hyperosmotic (raffinose) and normal conditions was investigated using confocal laser scanning microscopy. MDCK 1 cells were cultured on 1.12 cm2 filter support similarly to described above. The cells on filter support were then prepared as previously described,18 briefly, the cells were rinsed in HBSS (room temperature), fixed for 10 min in HBSS with 3% paraformaldehyde, and then permeabilized for 5 min in 0.1% Triton X-100 in PBS. The cells were stained with Alexa488− phalloidin (1:200) and were counterstained with 0.5 mM propidium iodide in PBS for 1 min. All preparation steps were performed at room temperature (20 °C). After washing in PBS, filters were mounted on coverslips and confocal imaging was performed on a Zeiss LSM 510 laser scanning confocal microscope, using a Zeiss plan apochromat ×63 oil-immersion objective with a numerical aperture of 1.4. Fluorophores were excited using an argon laser line at 488 nm and a HeNe laser line at 543 nm. The total protein level of MDCK I cells cultured under hyperosmotic (mannitol) and isosmotic conditions was investigated using Bradford protein assay. The cells were counted before the assay, and the determination was performed following the manufacture’s protocol. Data Treatment. In the uptake studies, the results are presented as uptake rates (pmol·cm−2·min−1). The concentration dependent uptake of ibuprofen is presented as uptake rates (pmol·cm−2·min−1), and the resulting concentration dependent uptake curves were fitted to the Michaelis−Menten kinetics (eq 1) using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). The total ibuprofen uptake was calculated using the fraction of [3H]-ibuprofen of the total amount of ibuprofen in the uptake buffer: uptake rate =

Vmax ·[S] K m + [S]

(1)

where [S] is the concentration of the substrate, Vmax the maximum velocity of the transporter, and Km is the concentration of the substrate, which gives 50% of Vmax. The cellular uptake rate (pmol·cm−2·min−1) of [3H]ibuprofen in the presence of increasing concentrations of (R)(−)-ibuprofen or (S)-(+)-ibuprofen was fitted to a fourparameter equation (eq 2), using GraphPad Prism 6.03 (GraphPad Software, Inc., La Jolla, CA, USA) to obtain an IC50 value: U = Umin +

Umax − Umin 1 + 10(log[I ] − log IC50)·nH −2

(2)

−1

U is the uptake rate (pmol·cm ·min ), [I] is the concentration of inhibitor (μM), Umax is the initial uptake rate (pmol·cm−2· min−1) ([I] = 0), Umax is the lowest measured concentration of ibuprofen, and nH is the Hill coefficient. C

DOI: 10.1021/acs.molpharmaceut.6b00330 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics The comparative CT method19 was used to compare mRNA expression levels in MDCK I cells. The fold change between control condition and hyperosmotic condition was calculated using eq 3: [(C T gene of interest − C T internal control)]control sample −

fold change = 2

[(C T gene of interest − C T internal control)]treated sample

(3)

where CT is the number of cycles it takes for the amplified cDNA to reach the threshold value. Statistical Analysis. The results shown are mean ± SEM of 3−5 experiments carried out on independent cell passages. The statistical analyses were performed using GraphPad Prism 6.03 (GraphPad Software, Inc., La Jolla, CA, USA). For the analysis of differences between means, a one-way ANOVA was performed followed by Tukey’s multiple comparison test. P > 0.05 indicates nonsignificance (ns). * Denotes levels of significance p < 0.05.

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RESULTS MDCK I Cells Cultured under Hyperosmotic Conditions Have Increased Saturable Ibuprofen Uptake. The cellular uptake rate of [3H]-ibuprofen at pH 7.4 was investigated in MDCK I cells cultured under iso-osmotic and hyperosmotic conditions (Figure 1). The uptake of [3H]-ibuprofen in MDCK I cells, cultured for the final 24 h in hyperosmotic (500 mOsm, mannitol) culture medium was approximately 5 times higher and could be significantly reduced by the presence of 1 mM unlabeled ibuprofen (Figure 1A). In contrast, the uptake of [3H]-ibuprofen in MDCK I cells cultured under isotonic culture conditions was not reduced in the presence of 1 mM unlabeled ibuprophen. The ability of different osmolytes to induce increased ibuprofen uptake was investigated (Figure 1B). The culture medium was supplemented with urea, NaCl, mannitol, or raffinose to a final osmolarity of 500 mOsm, and MDCK I cells were cultured with these solution for the last 24 h prior to the uptake study. Mannitol, NaCl, and raffinose significantly increased the uptake of [3H]-ibuprofen approximately 6−17-fold, and this uptake could be inhibited by unlabeled ibuprofen. In contrast, MDCK I cells cultured with urea did not show an increased [3H]ibuprofen uptake and the uptake was not significantly affected by unlabeled ibuprofen. The ability of NaCl and mannitol to increase [3H]-ibuprofen uptake was similar, whereas raffinose significantly increased [3H]-ibuprofen uptake compared to mannitol and NaCl. The uptake of ibuprofen at different extracellular concentrations was subsequently investigated in MDCK I cell cultured in normal medium and medium supplemented with mannitol or raffinose to a final osmolarity of 500 mOsm (Figure 1C). The uptake of ibuprofen in MDCK I cells was saturable under hyperosmotic conditions and could be described by Michaelis−Menten kinetics with Km values of 0.19 ± 0.05 and 0.37 ± 0.08 μM under mannitol and raffinose treated conditions, respectively. The Km values were not significantly different. The maximal uptake rate (Vmax) was 58.6 ± 4.2 and 233.1 ± 17.2 pmol·cm−2·min−1 under mannitol and raffinose treated conditions, respectively. The Vmax was significantly increased by the hyperosmotic conditions, and raffinose treatment significantly increased Vmax compared to mannitol treatment. Ibuprofen Uptake is pH-Dependent but Sodium and Chloride Independent. The uptake rate of [3H]-ibuprofen was investigated in MDCK I cells cultured under normal and hyperosmotic conditions in the absence or presence of

Figure 1. Cellular uptake of [3H]-ibuprofen in hyperosmotic treated MDCK I cells. (A) Cellular uptake rate of 1 μCi·mL−1 (0.2 μM) [3H]ibuprofen under iso-osmotic (300 mOsm) and hyperosmotic (500 mOsm by addition of mannitol) conditions in MDCK I cells in the absence or presence of 1 mM unlabeled ibuprofen. The buffer used was HBSS containing 10 mM HEPES adjusted to pH 7.4 at 37 °C. The uptake was measured for 10 min. Each column represents the mean ± SEM of four to five different cell passages (n = 4−5). One-way ANOVA with multiple comparisons Tukey’s test was used to determine the levels of statistical significance (*, p < 0.05). (B) Cellular uptake rate of 0.25 μCi·mL−1 (0.05 μM) [3H]-ibuprofen under iso-osmotic (300 mOsm) and hyperosmotic (500 mOsm by addition of mannitol, urea, NaCl, or D

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Molecular Pharmaceutics

(−)-ibuprofen in inhibiting the uptake rate of [3H]-ibuprofen. The ibuprofen uptake rate was not altered in the presence of 100 μM probencid, folic acid, furosemide, quercetin, fluvastatin, glutamic acid, or 1, 10, or 30 mM lactate (Figure 3C). Time-Dependent Up- and Down-Regulation of Ibuprofen Transport Caused by Hyperosmolarity. The ability of raffinose to upregulate [3H]-ibuprofen uptake rate in MDCK I cells was investigated over time (Figure 4A). The uptake rate of [3H]-ibuprofen was measured at different time points after the culture medium was switched from isosmotic culture medium (300 mOsm) to culture medium supplemented with raffinose (500 mOsm). Incubation in hyperosmotic medium decreased [3H]-ibuprofen uptake rate for the first 4 h, and after 6 h, the uptake rate was again similar to the uptake rate in isosmotic culture cells. After 22 h, the uptake rate of 3H-ibuprofen in hyperosmotic-treated cell was significantly higher than that in isosmotic treated cells, and from 30−48 h, the [3H]-ibuprofen uptake rate remained relatively constant. The upregulation of [3H]-ibuprofen uptake rate was thus relatively slow and not related to rapid regulation induced by osmolarity-related responses. The effect of changing the culture medium from hyperosmotic back to isosmotic medium was then investigated (Figure 4B). MDCK I cells were incubated for 24 h in raffinosesupplemented culture medium, after which the medium was changed to normal medium. After this change, the uptake rate of [3H]-ibuprofen still tended to increase, but after 18−24 h in normal medium, the uptake rate decreased. However, the uptake rate did not decrease to the level observed in MDCK I cells cultured in normal medium alone. The effect of incubating MDCK I cells with 50 μM ibuprofen, 50 μM acetylsalicylic acid, or 10 μM piroxicam, respectively, in the growth medium for the last 24 h before the uptake experiment on the uptake rate of [3 H]-ibuprofen was investigated. The presence of ibuprofen or piroxicam in the growth medium did not change the uptake rate of [3H]ibuprofen under normal conditions. However, when the growth medium was hyperosmotic (500 mOsm; raffinose), then the uptake rate of [3H]-ibuprofen was significantly lower if ibuprofen or pirocixam was present in the growth medium (Figure 5). Acetylsalicylic acid did not change the uptake rate of ibuprofen in MDCK I cells cultured under normal or hypertonic conditions. Hyperosmolarity Does Not Upregulate mRNA of Monocarboxylate or Organic Anion Transporters. To further verify that the ibuprofen transporter investigated was distinct from already suggested transporters, real-time PCR was performed. For comparison the taurine transporter, known to be upregulated by hyperosmotic conditions, was included. As shown in Figure 6A TauT mRNA was significantly upregulated 7.6- and 6.1-fold when MDCK I cell were cultured with raffinose and NaCl, respectively, compared to the isoosmotic control, whereas urea did not affect the expression of TauT mRNA. The mRNA of Mct11, Mct4, and Mct6 were all found to be expressed in MDCK1 cells, but the expression of the transcript were not altered in response to hyperosmolarity induced by culturing MDCKI cells with urea, NaCl, or raffinose (Figure 6B−D). The mRNA of Mct2, Oat1, and Oat3 were also investigated but were not expressed in MDCK I cells to a detectable mRNA level (data not shown). Morphology of MDCK I Cells Cultured under Isosmotic and Hyperosmotic Conditions. A series of vertical and horizontal scans of filter-grown MDCK I cell layers were performed using confocal laser scanning microscopy (Figure 7). The cell nuclei were labeled with propidium iodide, and the actin

Figure 1. continued raffinose) conditions in MDCK I cells in the absence or presence (indicated by +) of 250 μM unlabeled ibuprofen. The buffer used was HBSS containing 10 mM HEPES adjusted to pH 7.4 at 37 °C. The uptake was measured for 5 min. Each column represents the mean ± SEM of four to eight independent cell passages (n = 4−8). One-way ANOVA with multiple comparisons Tukey’s test was used to determine the levels of statistical significance (*, p < 0.05). (C) The uptake rate of ibuprofen in MDCK I cells cultured under hyperosmotic (500 mOsm by addition of mannitol or raffinose) or iso-osmotic (300 mOsm) conditions in the presence of increasing unlabeled ibuprofen. The buffer used was HBSS containing 10 mM HEPES adjusted to pH 7.4 at 37 °C. The uptake was measured for 5 min in raffinose-induced hyperosmolality and iso-osmotic conditions and 10 min mannitolinduced hyperosmotic conditions. The concentration of [3H]-ibuprofen was 0.125 (6 nM) or 0.25 μCi·mL−1 (12.5 nM) and the uptake time 5 min for raffinose-induced hyperosmotic and iso-osmotic conditions (300 mOsm), and 0.125−1 μCi·mL−1 (25−200 μM) and the uptake time 10 min for mannitol-induced hyperosmolality. Each value represents the mean ± SEM of four to seven different cell passages (n = 4−7). The solid lines show the fit of the resulting data to Michaelis− Menten like kinetics.

extracellular sodium or chloride (Figure 2). Regardless of the culture conditions, the uptake rate of [3H]-ibuprofen was not different in the presence or absence of extracellular sodium or chloride (Figure 2A). The uptake rate of [3H]-ibuprofen in MDCK I cells cultured in normal medium was significantly higher with an extracellular pH of 6.0 than with pH 7.4 (Figure 2B). However, at both pH 6.0 and 7.4, a surplus of unlabeled ibuprofen did not significantly decrease the uptake rate of [3H]ibuprofen. Under hyperosmotic culture conditions, the uptake rate of [3H]-ibuprofen was significantly increased by lowering the extracellular pH from 7.4 to 6.0. Under these conditions, the uptake rate of [3H]-ibuprofen could be inhibited by unlabeled ibuprofen at both pH 6.0 and pH 7.4. The uptake rate of [3H]ibuprofen was then investigate in MDCK I cells cultured under hyperosmotic conditions based on the addition of raffinose (Figure 2C,D). Again, the uptake rate of [3H]-ibuprofen was higher at pH 6.0 than at pH 7.4 (p < 0.05, n = 3−5), and the cellular [3H]-ibuprofen uptake rate in the presence of excess unlabeled ibuprofen was negligible. In the presence of nigericin, a H+/K+ antiporter, the uptake rate was reduced significantly at an extracellular pH of both 6.0 and 7.4. In the presence of nigericin, the uptake rate of [3H]-ibuprofen at pH 6.0 and 7.4 was not significantly different, and in the presence of nigericin, the uptake rate of [3H]-ibuprofen could be further reduced by unlabeled ibuprofen. (R)-(−)- and (S)-(+)-Ibuprofen Inhibits Racemic [3H]Ibuprofen Uptake Rate Differently. The uptake rate of [3H]ibuprofen, a racemic mixture, was measured in the presence of 0.5 μM (R)-(−) or (S)-(+)-ibuprofen to investigate the stereochemical selectivity (Figure 3A). At this concentration, (R)-(−)-ibuprofen did not significantly inhibit the [3H]ibuprofen uptake rate whereas (S)-(+)-ibuprofen significantly decreased the uptake rate of racemic [3H]-ibuprofen. The differences in the ability of (R)-(−)- and (S)-(+)-ibuprofen to inhibit [3H]-ibuprofen uptake rate was therefore further investigated (Figure 3B). The concentration-dependent inhibition of [3H]-ibuprofen uptake rate gave IC50 values for (R)-(−) and (S)-(+)-ibuprofen of 19 μM (Log IC50 1.39 ± 0.34) and 0.47 μM (Log IC50 −0.36 ± 0.41), respectively, which shows that (S)(+)-ibuprofen is more than 40 times more potent than (R)E

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Figure 2. Driving force of the cellular uptake of [3H]-ibuprofen in hyperosmotic treated MDCK I cells. (A) Cellular uptake rate of [3H]-ibuprofen in the presence or absence of sodium or chloride in MDCK I cells cultured under iso- (300 mOsm) and hyperosmotic (500 mOsm by addition mannitol or raffinose) conditions. The buffers contained 10 mM HEPES adjusted to pH 7.4 at 37 °C. For mannitol induced hyperosmolality, the [3H]-ibuprofen concentration was 0.5 μCi·mL−1 (0.1 μM) and the uptake time 10 min, whereas for raffinose induced hyperosmolality, the [3H]-ibuprofen concentration was 0.5 μCi·mL−1 (0.025 μM) and the uptake time 5 min. Each column represents the mean ± SEM of three to five different passages (n = 3−5). (B) The proton dependent uptake rate of [3H]-ibuprofen uptake in MDCK I cells cultured under iso- (300 mOsm) and hyperosmotic (500 mOsm by addition mannitol) conditions. The uptake was in the presence and absence of 250 μM unlabeled ibuprofen. The uptake time was 10 min. The buffer used was HBSS containing either 10 mM HEPES or 10 mM MES adjusted to pH 7.4 or 6.0, respectively, at 37 °C. Each column represents the mean ± SEM of three to six different passages (n = 3−6). One-way ANOVA with multiple comparisons Tukey’s test was used to determine the levels of statistical significance (*, p < 0.05). (C) The uptake rate of [3H]-ibuprofen at pH 7.4 in MDCK I cells cultured under hyperosmotic (500 mOsm by addition raffinose) conditions. The uptake was determined in the absence and presence of 100 μM unlabeled ibuprofen, 20 μM nigericin, or 100 μM ibuprofen and 20 μM nigericin, respectively. The uptake time was 5 min. The buffer used was HBSS containing 10 mM HEPES adjusted to pH 7.4 at 37 °C. Each column represents the mean ± SEM of four to five different passages (n = 4−5). One-way ANOVA with multiple comparisons Tukey’s test was used to determine the levels of statistical significance from the control (*, p < 0.05). (D) The uptake rate of [3H]-ibuprofen uptake at pH 6.0 in MDCK I cells cultured under hyperosmotic (500 mOsm by addition raffinose) conditions. The uptake was determined in the absence (control) or presence of 100 μM unlabeled ibuprofen, 20 μM nigericin or 100 μM ibuprofen, and 20 μM nigericin, respectively. The buffer used was HBSS containing 10 mM MES adjusted to pH 6.0 at 37 °C. The uptake time was 5 min. Each column represents the mean ± SEM of three different passages (n = 3). One-way ANOVA with multiple comparisons Tukey’s test was used to determine the levels of statistical significance from the control (*, p < 0.05).

was labeled with Alexa 488-conjugated phalloidin. The actin skeleton lies just beneath the cell membrane and thereby visualizes the outline of the cells. The vertical scans showed that

both MDCK I cells cultured under normal and hyperosmotic conditions formed a monolayer, with cell heights around 10 μM. Cells cultured under hyperosmotic conditions appeared slightly F

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in MDCK I cells cultured under hyperosmotic (500 mOsm, mannitol) conditions. The buffers contained 10 mM HEPES adjusted to pH 7.4 at 37 °C. The [3H]-ibuprofen concentration was 0.5 μCi·mL−1 (0.1 μM), and the uptake time was 10 min. Each column represents the mean ± SEM of three to five different passages (n = 3−5).

Figure 4. Effect of the exposure time of hyperosmotic medium on the uptake of [3H]-ibuprofen in MDCK I cells. (A) Uptake of 0.6 μCi·mL−1 [3H]-ibuprofen (0.03 μM) in MDCK I cells cultured under hyperosmotic (500 mOsm by the addition of raffinose) conditions at different time points. The cultivation time in hyperosmotic medium varied from 0 to 48 h. The buffer used was HBSS containing 10 mM HEPES adjusted to pH 7.4 at 37 °C. The uptake time was 5 min. Each value represents the mean ± SEM of three to six different passages (n = 3−6). (B) Uptake of 0.6 μCi·mL−1 [3H]-ibuprofen (0.03 μM) in MDCK I cells cultured under hyperosmotic (500 mOsm by the addition of raffinose) conditions for 24 h and switched back to isosmotic (300 mOsm) cultivation conditions. The exposure time of isosmotic conditions after the switch from hyper to isosmotic was 2−24 h. The buffer used was HBSS containing 10 mM HEPES adjusted to pH 7.4 at 37 °C. The uptake time was 5 min. Each value represents the mean ± SEM of four to six different passages (n = 4−6).

Figure 3. Stereoselective inhibitory effect of ibuprofen enantiomers on the uptake of [3H]-ibuprofen in MDCK I cells cultured under hyperosmotic conditions. (A) Uptake of 0.6 μCi·mL−1 [3H]-ibuprofen (0.03 μM) in MDCK I cells cultured under hyperosmotic (500 mOsm by addition raffinose) conditions in the absence (control) or presence of each enantiomer of ibuprofen at 0.5 μM. The buffer used was HBSS containing 10 mM HEPES adjusted to pH 7.4 at 37 °C. The uptake time was 5 min. Each column represents the mean ± SEM of four different passages (n = 4). One-way ANOVA with multiple comparisons. Tukey’s test was used to determine the levels of statistical significance (*, p < 0.05). (B) Concentration−inhibition curve for 0.6 μCi·mL−1 [3H]ibuprofen (0.03 μM) uptake in MDCK I cells cultured under hyperosmotic (500 mOsm by addition raffinose) conditions in the presence of (R)-(−) and (S)-(+)-ibuprofen at concentrations in the range of 0.5−100 μM and 0.001−2.5 μM, respectively. The buffer used was HBSS containing 10 mM HEPES adjusted to pH 7.4 at 37 °C. The uptake time was 5 min. Each value represents the mean ± SEM of three to six different passages (n = 3−6). The solid lines show the fit of the resulting data to eq 2. (C) Cellular uptake rate of [3H]-ibuprofen in the presence or absence of various inhibitors of MCT and OAT transporters

taller, but no apparent morphological differences between the two conditions was evident. The number of cells per well was counted after culturing in normal medium and after 24 h in mannitol-supplemented medium. The number of cells per well were not significantly different between the two conditions: 1.75 × 106 ± 3.1 × 105 cells per well in normal medium versus 1.55 × 106 ± 3.6 × 105 cells per well in hyperosmotic medium (p > 0.05, n = 3). Under similar conditions, the protein amount was measured. In cells cultured under normal conditions, the protein amount was 28.3 ± 4.0 μg per well and under hyperosmotic culture conditions 23.1 ± 3.3 μg per well. Hyperosmotic culture G

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transporter-mediated uptake, because passive transport processes are not saturable or inhibitable. Other nonpermeant osmolytes such as NaCl and raffinose also induced increased ibuprofen uptake rate when added to the culture medium for 24 h prior to the uptake experiment. After hyperosmotic treatment, the uptake rate of ibuprofen was saturable and could be described by Michaelis−Menten like kinetics as a further evidence for the presence of an ibuprofen transporter. Transporters are often stereoselective in terms of substrate or inhibitor recognition. Here, both the stereochemical forms of ibuprofen inhibited the ibuprofen uptake rate in a concentration dependent manner but with different affinities. This is a clear indication of the involvement of a transport protein in the movement of ibuprofen across the cell membrane. The pharmacological active (S)(+)-ibuprofen was 40 times more potent than (R)-(−)-ibuprofen in inhibiting [3H]-ibuprofen uptake rate as evaluated by their IC50 values. It has previously been shown that the stereoisomers of ibuprofen have different abilities to inhibit lactate uptake in CHO cells, presumably via MCT1.13 Another feature of transporters (specifically carriers of the solute carrier family, SLC) is their frequent reliance on other cosubstrates to generate the driving force for substrate uptake, using either ions or organic solutes via a symport or antiport process. Ibuprofen uptake was not dependent on symport of sodium or chloride ions after induction by neither mannitol nor raffinose. Investigations of the effect of extracellular proton concentrations on ibuprofen transport was complicated by the fact that lowering pH increases the fraction of the neutral form of ibuprofen, which has higher membrane permeability compared to the anionic species of ibuprofen.3 Here, the uptake rate of ibuprofen was increased by increasing the extracellular proton concentration from pH 7.4 to 6.0. However, the uptake could be almost completely inhibited by unlabeled ibuprofen. After preincubation with nigericin, a molecule acting as a proton pump equalizing the intracellular and extracellular proton concentration, the uptake rate of ibuprofen was reduced. At pH 6.0, the uptake rate in the presence of nigericin was 24% of the control uptake rate while it was 69% at pH 7.4. The uptake of ibuprofen in nigericin treated cell could also be inhibited by unlabeled ibuprofen. This suggests that the ibuprofen transporter is capable of transporting anionic charged ibuprofen without a transmembrane proton gradient. It is not possible based on this to exclude the possibility that protons may be part of the translocation cycle. The uptake of ibuprofen was not inhibited by different concentrations of lactate, a substrate of MCTs,13−15 and 100 μM of quercetin did not inhibit ibuprofen uptake. Quercetin has affinities for MCT1 and MCT2 of 14 and 5 μM, respectively,21 and it thus seems unlikely that the ibuprofen transporter studied here is one of the MCTs. Further support for this interpretation is the lack of hyperosmotic induced changes in the steady-state mRNA expression of Mct1, Mct4, and Mct6, and that Mct2 mRNA expression could not be detected by real-time PCR. Because the ibuprofen uptake is highly increased under hyperosmotic conditions, while no changes are present for Mct1, Mct4, and Mct6 mRNA expression, these transporters are not likely to cause the increased ibuprofen uptake. This point to the fact that Mct1, Mct4, and Mct6 are not regulated at the transcriptional level, however, osmolarity could increase the membrane density of these transporter via recruitment of already synthesized transporters from intracellular pools. Khamdang et al. investigated the uptake of 0.5 μM ibuprofen in hOAT1 and hOAT3 transfected S2 cells, where the uptake was increased 1.4and 1.7-fold compared to the corresponding mock-transfected S2

Figure 5. The effect on [3H]-ibuprofen uptake in MDCK I cells in the presence of ibuprofen, acetylsalicylic acid, or piroxicam. Cellular uptake rate of [3H]-ibuprofen 0.6 μCi·mL−1 in MDCK I cells cultured for the last 24 h under iso- (300 mOsm) and hyperosmotic (500 mOsm by addition raffinose) conditions in the presence of either 50 μM ibuprofen, 50 μM acetylsalicylic acid, or 10 μM piroxicam, respectively. The buffers contained 10 mM HEPES adjusted to pH 7.4 at 37 °C. Each column represents the mean ± SEM of four to five different passages (n = 4−5). One-way ANOVA with multiple comparisons Tukey’s test was used to determine the levels of statistical significance (*, p < 0.05).

conditions thus did neither change the amount of cells per well nor the protein amount per well.

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DISCUSSION Ibuprofen has been identified as an inhibitor of substrate transport via several transporters; however, so far the evidence of an actual ibuprofen transporter has been limited to a few indications.7,12 In the present study, several novel findings were revealed studying ibuprofen uptake in renal MDCK I cells such that (i) a so far uncharacterized ibuprofen transporter was identified at the functional level, (ii) this transporter was upregulated by hyperosmotic culture conditions, and (iii) the upregulation was a long-term effect of osmolarity and was partially reversible when cells were returned to isosmotic culture conditions. Ibuprofen Uptake in MDCK I Cells Is TransporterMediated. Ibuprofen uptake in MDCK I cells showed several characteristics supporting the conclusion that a carrier was involved in facilitating the cellular uptake. The first indication of an ibuprofen transporter came from the increased ibuprofen uptake rate in MDCK I cells cultured in hyperosmotic culture medium where mannitol was used to increase the osmolarity from 300 to 500 mOsm. The cellular effect of hyperosmolarity is often studied under wide changes in absolute osmotic increases (30−1000 mOsm, as reviewed in ref 20 for different incubation times (min to days) and in many cell lines.20 Under hyperosmotic conditions, the uptake rate of ibuprofen could be inhibited by unlabeled ibuprofen, which is indicative of H

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Figure 6. Relative mRNA expression of the transporters TauT (A), Mct1 (B), Mct4 (C), and Mct6 (D) in iso-osmotic (300mOsm) and hyperosmotic (urea, NaCl, or raffinose, 500 mOsm) treated MDCK I cells. Control is expression under isosmotic culture condition. The expression was analyzed with real-time PCR and normalized to the expression of Hprt mRNA. Mean ± SEM ** = P < 0.01. n = 4−5 passages.

cells.12 In the study, it was concluded that hOAT1 and hOAT3 transports ibuprofen, although regulatory guidelines suggest that the increase compared to empty vector control should be more than two.22 In our study, inhibitors of OAT1-mediated transport, probenecid and furosemide, did not inhibit ibuprofen uptake at a concentration of 100 μM. Considering that probenecid has affinities for OAT1 of 4.3−12.5 μM and furosemide 14−20 μM,23 it seems reasonable to conclude that the observed ibuprofen transport in MDCK 1 cells is not via OAT1. Likewise, 100 μM of furosemide, folate, fluvastatin, and probenecid is well above the affinities of these drugs for OAT3,23 and therefore it is unlikely that OAT3 is the transporter responsible for ibuprofen uptake in hyperosmotic treated MDCK 1 cells, especially considering that Oat1 and Oat3 mRNA expression could not be detected under iso-osmotic or hyperosmotic culture conditions. It is well-known that a number of transporters are upregulated in renal cells as a response to increased osmolarity in order to facility cellular uptake of organic osmolytes.20 These transporters include the taurine transporter, TauT, the betaine/GABA transporter, BGT1, and the sodium-coupled myo-inositol transporter, SMIT1.20 While we show here that TauT is upregulated by hyperosmolarity, TauT, BGT1, and SMIT1 are symporters transporting their substrates together with sodium ions, and transport via TauT and BGT1 is furthermore chloridecoupled. The ibuprofen transporter we identified in the present work is not dependent on sodium or chloride ions, which is why it is unlikely that we are studying TauT, BGT1, or SMIT1. It is therefore not clear which gene is responsible for the ibuprofen uptake transporter or why osmolarity increases the functionality of this carrier.

Upregulation of the Ibuprofen Transporter. The ibuprofen carrier was upregulated in response to the extracellular presence of NaCl, mannitol, and raffinose but not urea. It required several hours (∼20) to get a functional upregulation expressed, and after the switch back to isosmotic medium, the functionality decreased, though not completely, over several hours. This suggests that upregulation is an adaptive response rather than an immediate response to hyperosmolarity. As opposed to mannitol, NaCl, and raffinose, urea is a membrane permeable compound. It therefore seems likely that the osmotic gradient across the cell membrane rather than total extracellular osmolarity or a specific osmolyte effect induced the transporter responsible for ibuprofen transport. Mannitol is a monosaccharide and raffinose is a trisaccharide, and the ability to cross the cell membrane is likely lower for raffinose than for mannitol. The maximal transport rate was higher after raffinose treatment than mannitol treatment, which was likely due to the minor raffinose membrane permeability and, hence, a higher osmotic gradient. The regulatory pattern observed for the ibuprofen carrier was quite similar to the raffinose-induced regulation of BGT1. In MDCK cells, betaine uptake was increased after incubation with medium supplemented with raffinose to 500 mOsm and reached a peak 24−30 h after the switch from isotonic to hyperosmotic medium.24 After switching from hyperosmotic to isotonic medium, the BGT1 function was downregulated.24 The underlying molecular mechanism was increased and decreased transcription of BGT1 mRNA, respectively.24 In the present study, we do not presently know the underlying molecular reason for the increased ibuprofen uptake, which complicates further molecular studies. However, it seems likely that the increased uptake rate was due to increased I

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conditions. Ibuprofen is not a physiologically relevant osmolyte, which raises the question about what endogenous substrate the upregulated transporter has and if the upregulation is an element in accumulating organic osmolytes. In conclusion, we here present evidence for the presence of a so far uncharacterized ibuprofen transporter in MDCK I cells, which is upregulated by hyperosmotic culture conditions. Previously suggested ibuprofen transporters of the monocarboxylate and organic anion families were not upregulated at the mRNA level in response to increased osmolarity. The transporter recognizes ibuprofen in a stereospecific manner, with the pharmacologically active (S)-(+)-ibuprofen having the highest affinity, and the transporter is not dependent on symport of sodium or chloride ions. The transporter display characteristics distinct from OAT transporters, which are so far, to the best of our knowledge, the only transporters that have been proposed to accept ibuprofen as a substrate.

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ASSOCIATED CONTENT

S Supporting Information *

Figure 7. The morphological changes of MDCK I cells cultured under hyperosmotic (500 mOsm by addition of raffinose) conditions. (A) Confocal laser scanning vertical section images of MDCK 1 monolayers stained with propidium iodide and Alexa 488-conjugated phalloidin. Two separate filters, one cultured in normal medium (left) and one cultured for 24 h in hyperosmotic medium (raffinose) (right), were aligned on a coverslip to allow a direct comparison of the fluorescence from the two filters at the same gain settings. The propidium iodide signal is shown in red, and the signal from the Alexa 488-conjugated phalloidin is shown in green. (B) Horizontal section through MDCK 1 cell monolayers (apical membrane facing upward) cultured in normal medium. (C) Horizontal section through MDCK 1 cell monolayers (apical membrane facing upward) cultured for 24 h in hyperosmotic medium (raffinose). The image shown is representative for experiments performed in three independent cell passages. The white bar is 10 μm.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b00330.

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Overview of primers for real-time PCR used for canine genes (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: +45 6550 9427. E-mail: [email protected]. Present Address ∥

(R.H.) Drug Product Development, Janssens Research and Development, Johnson & Johnson, Turnhoutseweg 30, 2430 Beerse, Belgium

expression of mRNA coding for the relevant transporter followed by insertion in the plasma membrane rather than short-term membrane insertion of already synthesized transporters from intracellular pools or post-translation modification altering transporter kinetic parameters, similar to what has previously been reported for BGT1 and TauT.24−27 A key element in the upregulation of transporters mediating cellular accumulation of organic osmolytes is the transcriptional activator TonEBP/ OREBP, also named NFAT5. Generally, it has been shown that TonEBP/OREBP is involved in upregulation of SMIT1, TauT, and BGT1 in response to hyperosmotic condition (as reviewed in ref 20). In MDCK cells, it has been reported that hypertonicity increases TonEBP/OREBP mRNA expression after hyperosmotic stress.28 In the human corneal epithelial cell line, RCB2280, 1 h of incubation in hyperosmotic medium increases the amount of NFAT5 in the nucleus and the expression of BGT1 mRNA.29 Interestingly, it was shown that 50 μM diclofenac reduced the cell damage (measured by an MTT assay) after 24 h incubation in 600 mOsm medium, whereas ibuprofen and aspirin did not.29 Although ibuprofen and aspirin were not investigated further, diclofenac increased NFAT5 expression under hyperosmotic condition and increased BGT1 mRNA expression in a COX independent manner.29 In the present study, 24 h incubation with 50 μM ibuprofen or piroxicam in a hyperosmotic medium reduced ibuprofen uptake rate compared to hyperosmolarity alone, whereas aspirin had no effect. Furthermore, ibuprofen, piroxicam, or aspirin alone did not induce upregulation of the ibuprofen transport under iso-osmotic

Author Contributions

C.U.N. contributed to the design of the study, performed data collection and analysis, and data interpretation, and drafting of the manuscript. R.N.R. contributed to the design of the study, performed data collection, analysis, and data interpretation, and drafting of the manuscript. J.M. contributed to the design of the study and performed data collection and analysis and data interpretation. B.N. contributed to the design of the study and performed data collection and analysis and data interpretation. C.L. contributed to the design of the study and performed data collection and analysis and data interpretation. R.H. contributed to the design of the study, data interpretation, and drafting of the manuscript. M.K.N. contributed to the design of the study, data interpretation, and drafting of the manuscript. All authors approved the final manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The cell culture facility at Department of Pharmacy, University of Copenhagen, is acknowledged for cell culturing (Maria Pedersen). The effort of Birgitte Eltong in generating CLSM images is highly acknowledged. The experimental part of the present work was performed during Carsten Uhd Nielsen’s employment at University of Copenhagen. J

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ABBREVIATIONS USED BGT1, betaine/GABA transporter; CHO, Chinese hamster ovary; HBSS, Hank’s Buffered Saline Solution; HEPES, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; Hprt, hypoxanthine-guanine phosphoribosyltransferase; Km, Michaelis constant; MCT, monocarboxylate transporter; MDCK, Madin− Darby canine kidney cells; MES, 2-(N-morpholino)ethanesulfonic acid; OATs, organic anion transporters; OCTs, organic cation transporters; SMCT1, sodium-coupled myoinositol transporter 1; TauT, taurine transporter; Vmax, maximum transport rate

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DOI: 10.1021/acs.molpharmaceut.6b00330 Mol. Pharmaceutics XXXX, XXX, XXX−XXX