0090-9556/09/3707-1404–1410$20.00
`DRUG METABOLISM AND DISPOSITION
`Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
`DMD 37:1404–1410, 2009
`
`Vol. 37, No. 7
`27169/3486872
`Printed in U.S.A.
`
`The Drug of Abuse ␥-Hydroxybutyrate Is a Substrate for Sodium-
`Coupled Monocarboxylate Transporter (SMCT) 1 (SLC5A8):
`Characterization of SMCT-Mediated Uptake and Inhibition
`
`Dapeng Cui and Marilyn E. Morris
`
`Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State
`University of New York, Amherst, New York
`
`Received February 18, 2009; accepted April 20, 2009
`
`ABSTRACT:
`
`␥-Hydroxybutyric acid (GHB), a drug of abuse, is a substrate of
`monocarboxylate transporters (MCTs). Sodium-coupled monocar-
`boxylate transporter 1 (SMCT1; SLC5A8) is expressed in kidney,
`thyroid gland, neurons, and intestinal tract and exhibits substrate
`specificity similar to that of the proton-dependent MCT (SLC16A)
`family. The role of SMCT1 in GHB disposition has not been deter-
`mined. In this study we characterized the driving force, transport
`kinetics, and inhibitors of GHB uptake, as well as expression of
`SMCT and MCT isoforms, in rat thyroid follicular (FRTL-5) cells.
`GHB, as well as the monocarboxylates butyrate and D-lactate,
`exhibited sodium-dependent uptake at pH 7.4, which could be
`described with a simple Michaelis-Menten equation plus a diffu-
`sional component [Km 0.68 ⴞ 0.30 mM, Vmax 3.50 ⴞ 1.58 nmol 䡠
`mgⴚ1 䡠 minⴚ1, and diffusional clearance (P) 0.25 ⴞ 0.08 ␮l 䡠 mgⴚ1 䡠
`minⴚ1]. In the absence of sodium, GHB uptake was significantly
`
`increased at lower pH, suggesting proton-gradient dependent
`transport. Reverse transcriptase-polymerase chain reaction and
`Western analyses demonstrated the expression of SMCT1, MCT1,
`and MCT2 in FRTL-5 cells, supporting the activity results. Sodium-
`dependent GHB uptake in FRTL-5 cells was inhibited by MCT
`substrates (D-lactate, L-lactate, pyruvate, and butyrate), nonsteroi-
`dal anti-inflammatory drugs (ibuprofen, ketoprofen, and naproxen),
`and probenecid. IC50 values for L-lactate, ibuprofen, ketoprofen,
`and probenecid were 101, 31.6, 64.4, and 380 ␮M, respectively.
`All four inhibitors also significantly inhibited GHB uptake in rat
`MCT1 gene-transfected MDA/MB231 cells, suggesting they are not
`specific for SMCT1. Luteolin and ␣-cyano-4-hydroxycinnimate
`represent specific proton-dependent MCT inhibitors. Our findings
`indicate that GHB is a substrate for both sodium- and proton-
`dependent MCTs and identified specific inhibitors of MCTs.
`
`Two members of the sodium-coupled monocarboxylate transporter
`family (SMCT) have been cloned to date, the high-affinity transporter
`SMCT1 (SLC5A8) and the low-affinity SMCT2 (SLC5A12) (Rodri-
`guez et al., 2002; Srinivas et al., 2005). The SLC5A8 gene was
`originally identified from a kidney cDNA library as a close structural
`relative of human Na⫹/I⫺ symporter (SLC5A5) (Rodriguez et al.,
`2002). SMCT1 protein has been detected in kidney, intestine, salivary
`gland, thyroid gland, brain, and retina (Ganapathy et al., 2008). Less
`is known about SMCT2. mRNA expression of SMCT2 was detected
`in kidney, small intestine, and skeletal muscle and to a lesser extent in
`brain and retina. Functional characterization of SMCT2 suggested
`substrate specificity similar to that of SMCT1. However, the affinities
`of SMCT2 for monocarboxylate substrates are approximately 35- to
`80-fold lower than those of SMCT1 (Srinivas et al., 2005).
`SMCT1 mediates transport of monocarboxylic acids such as lactate,
`
`This work was supported by the National Institutes of Health National Institute
`on Drug Abuse [Grant DA023223].
`Article, publication date, and citation information can be found at
`http://dmd.aspetjournals.org.
`doi:10.1124/dmd.109.027169.
`
`pyruvate, propionate, butyrate, nicotinate, and short-chain fatty acids.
`Substrates of SMCT1 are similar to those of MCTs, a proton-coupled
`monocarboxylate transporter family (Gopal et al., 2005; Spanier and
`Drewes, 2007; Morris and Felmlee, 2008). The physiological function
`of SMCT1 may relate to maintaining homeostasis of short monocar-
`boxylates, but clear evidence of its function is currently lacking. In the
`kidney, SMCT1 is expressed in the apical membrane of tubular
`epithelial cells and is mainly limited to the S3 section of the proximal
`tubule. It was hypothesized that SMCT1 is involved in renal reab-
`sorption of lactate and pyruvate (Gopal et al., 2007b; Ganapathy et al.,
`2008), because sodium-dependent transport of these monocarboxy-
`lates has been observed in the kidney (Barac-Nieto et al., 1980;
`Mengual et al., 1989). Moreover, SLC5A8-deficient or knockout mice
`exhibited increased urinary excretion of lactate (Frank et al., 2008). In
`the brain, SMCT1 exhibits a neuron-specific distribution and may
`mediate cellular uptake of lactate and ketone bodies, the primary
`energy substrates of neurons (Martin et al., 2006). Besides the pos-
`tulated physiological functions, several reports suggested a tumor-
`suppressing role for SMCT1. High frequency of aberrant methylation
`or down-regulation of the SLC5A8 gene has been observed in human
`colon cancer, papillary thyroid carcinomas, pancreatic cancer, prostate
`
`ABBREVIATIONS: SMCT, sodium-dependent monocarboxylate transporter; MCT, monocarboxylate transporter; GHB, ␥-hydroxybutyrate; TSH,
`thyroid-stimulating hormone; BHB, ␤-hydroxybutyrate; CHC, ␣-cyano-4-hydroxycinnimate; DIDS, 4,4⬘-diisothiocyanatostilbene-2,2⬘-disulfonic
`acid; RT, reverse transcriptase; PCR, polymerase chain reaction; MES, 2-(N-morpholino)ethanesulfonic acid; ANOVA, analysis of variance; AIC,
`Akaike information criterion; NSAID, nonsteroidal anti-inflammatory drug.
`
`1404
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`Page 1 of 7
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`JAZZ EXHIBIT 2007
`Amneal Pharms. LLC (Petitioner) v. Jazz Pharms. Ireland LTD. (Patent Owner)
`Case IPR2016-00546
`
`

`
`SODIUM-DEPENDENT MCT TRANSPORT OF GHB
`
`1405
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`tumor, acute myeloid leukemia, and glioma formation (Ganapathy et
`al., 2008; Park et al., 2008).
`The role of SMCT1 in drug transport has not been determined. The
`expression pattern in kidney and intestine indicates that SMCT1 may
`play a role in the oral bioavailability and renal reabsorption of mono-
`carboxylate drugs. Studies using Xenopus laevis oocytes expressing
`human SLC5A8 showed that benzoate, salicylate, and 5-aminosalicy-
`late were transported by SMCT1 (Gopal et al., 2007a). In the present
`investigation, we characterized transport of ␥-hydroxybutyrate (GHB)
`in rat thyroid follicular FRTL-5 cells. GHB is an endogenous short-
`chain fatty acid formed from GABA and is present in the brain, heart,
`kidney, liver, lung, muscle, and gastrointestinal tract (Maitre, 1997;
`Tedeschi et al., 2003). The therapeutic use of GHB includes treatment
`of the sleep disorder narcolepsy with cataplexy (Mamelak et al., 1986)
`and alcohol withdrawal syndrome (Poldrugo and Addolorato, 1999).
`GHB is also a recreational drug, abused because of its euphoric effects
`(Wong et al., 2004). An overdose of GHB can lead to adverse effects
`such as seizures, dizziness, nausea, vomiting, coma, and even death
`(Mason and Kerns, 2002). GHB exhibits nonlinear pharmacokinetics
`in humans (Palatini et al., 1993) and rats (Lettieri and Fung, 1979),
`and the nonlinearity is related to capacity-limited metabolism (Ferrara
`et al., 1992), saturable absorption (Arena and Fung, 1980), and non-
`linear renal clearance (Morris et al., 2005). Our previous studies have
`demonstrated that MCT1 mediates GHB transport in rat kidney mem-
`brane vesicles,
`in the human kidney cell
`line HK-2, and in rat
`MCT1-transfected MDA-MB231 cells (Wang et al., 2006). In vivo
`studies have suggested that saturation of the MCT1-mediated renal
`reabsorption of GHB contributes to the increase of renal clearance at
`high GHB doses (Morris et al., 2005) and may represent a novel
`clinical treatment for GHB intoxification (Morris et al., 2005; Wang
`and Morris, 2007). However, the role of SMCTs in the disposition of
`GHB is largely unknown.
`The similarity of substrates of SMCTs and MCTs suggests that
`GHB may be a substrate of SMCT1. In this investigation, SMCT1-
`mediated transport of GHB and other MCT substrates, D-lactate and
`butyrate, was characterized using FRTL-5 cells, a rat thyroid-derived
`cell line that maintains thyrocyte functions such as hormonal respon-
`siveness, iodide uptake, and thyroglobulin synthesis (Ambesi-Impi-
`ombato et al., 1980). This cell line was chosen because expression of
`SMCT1 and the sodium-dependent transport of nicotinate have been
`observed in FRTL-5 cells (Paroder et al., 2006). Our overall goal is to
`characterize the SMCT-mediated transport of GHB and to identify
`specific transport inhibitors to develop strategies to treat GHB over-
`doses through inhibition of SMCT1-mediated transport in the body.
`The objectives of the study were 1) to evaluate the expression of
`SMCT1, 2) to examine the effect of sodium on the transport of GHB,
`as well as the monocarboxylate substrates D-lactate and butyrate, 3) to
`characterize the driving forces and concentration-dependent kinetics
`of GHB uptake, and 4) to identify potential inhibitors of SMCT1 in
`FRTL-5 cells.
`
`Materials and Methods
`
`Materials. Coon’s modified Ham’s F-12 medium and calf bovine serum
`were obtained from American Type Culture Collection (Manassas, VA).
`Dulbecco’s modified Eagle’s medium, trypsin-EDTA, fetal bovine serum,
`Geneticin (G-418), penicillin/streptomycin, and collagenase were obtained
`from Invitrogen (Carlsbad, CA). Bovine thyrotropic hormone (TSH), insulin,
`hydrocortisone, transferrin, somatostatin, glycyl-L-histidyl-L-lysyl acetate, D-
`lactate, L-lactate, sodium GHB, butyrate, pyruvate, DL-␤-hydroxybutyrate
`naproxen, ␣-cyano-4-hydroxycinnimate
`(BHB),
`ketoprofen,
`ibuprofen,
`(CHC), 3⬘,4⬘,5,7-tetrahydroxyflavone (luteolin), phloretin, probenecid, 4,4⬘-
`diisothiocyanatostilbene-2,2⬘-disulfonic acid (DIDS), HEPES, and MES were
`obtained from Sigma-Aldrich (St. Louis, MO). [2,3-3H]GHB (specific activity
`
`50 Ci/mmol), [2,3-3H]D-lactate (specific activity 20 Ci/mmol), and [1-14C]bu-
`tyrate (specific activity 56 mCi/mmol) were purchased from American Radio-
`labeled Chemicals (St. Louis, MO). Biodegradable counting scintillate was
`obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK).
`Cell Culture. FRTL-5 cells (American Type Culture Collection) were
`cultured as described previously (Beguinot et al., 1987). In brief, cells were
`grown in Coon’s modified Ham’s F-12 medium supplemented with 5% calf
`bovine serum and a mixture of hormones consisting of 10 mU/ml TSH, 0.01
`mg/ml insulin, 10 nM hydrocortisone, 5 ␮g/ml transferrin, 10 ng/ml soma-
`tostatin, and 10 ng/ml glycyl-L-histidyl-L-lysyl acetate. Cells were incubated at
`37°C in a humidified atmosphere with 5% CO2/95% air. Culture medium was
`changed every 3 to 4 days, and cells were passaged biweekly using 0.05%
`trypsin-EDTA with 20 U/ml collagenase. For uptake studies, cells were seeded
`in 35 mm2 plastic dishes or six-well plates and maintained for 1 week in the
`culture medium free of TSH. Rat MCT1 gene-transfected MDA-MB231 cells
`and mock cells were kindly provided by Professors I. Tamai and A. Tsuji
`(Kanazawa University, Kanazawa City, Japan) and cultured as described
`previously (Wang et al., 2006). In brief, cells were cultured at 37°C with 5%
`CO2/95% air in Dulbecco’s modified Eagle’s medium with addition of 10%
`fetal bovine serum, 0.5 mg/ml Geneticin, 100 units of penicillin, and 100
`␮g/ml streptomycin. Culture medium was changed every 2 to 3 days, and cells
`were passaged weekly. Two or 3 days before the uptake studies, cells were
`seeded in six-well plates with a density of 1 ⫻ 105 cells/well.
`Cellular Uptake Study. Culture medium was removed, and cells were
`washed three times with uptake buffer (137 mM NaCl, 5.4 mM KCl, 2.8 mM
`䡠 6H2O, and 10 mM HEPES, pH 7.4). One milliliter of
`CaCl2, 1.2 mM MgCl2
`uptake buffer containing [3H]GHB, [3H]D-lactate, or [14C]butyrate was added
`to the dishes. For the time course study, the cells were incubated at room
`temperature for 0.5, 1, 3, 5, 10, 30, and 60 min. For the pH-dependent study,
`the cells were incubated with uptake buffer with different pH values (pH 5.5,
`6.0, 6.5, 7.0, and 7.4), whereas for the sodium-dependent study, the cells were
`incubated with sodium buffer (137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2, 1.2
`䡠 6H2O, and 10 mM HEPES, pH 7.4) or sodium-free buffer (137
`mM MgCl2
`mM N-methyl-D-glucamine, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10
`mM HEPES, pH 7.4). Inhibitors examined in the cellular uptake studies
`included D-lactate, L-lactate, butyrate, pyruvate, BHB, ketoprofen, ibuprofen,
`naproxen, CHC, luteolin, phloretin, probenecid, and DIDS. The uptake was
`stopped by aspirating the buffer and washing three times with ice-cold stop
`䡠 6H2O,
`buffer (137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgCl2
`and 10 mM HEPES, pH 7.4). The cells were lysed in 1 ml of lysis buffer (0.3
`N NaOH and 1% SDS) for 2 h. Radioactivity was determined by mixing 3 ml
`of scintillation liquid with 400 ␮l of lysed sample and counting with a liquid
`scintillation counter (1900 CA, Tri-Carb liquid scintillation analyzer;
`PerkinElmer Life and Analytical Sciences, Waltham, MA). Protein concentra-
`tions were determined by the bicinchoninic acid protein assay with bovine
`serum albumin as the protein standard. The results were normalized for the
`protein content of the cell lysate.
`RT-PCR. Total RNA was isolated from cells using the SV Total RNA
`Isolation System (Promega, Madison, WI). First-strand cDNA was synthesized
`from 10 ␮g of total RNA by SuperScript II reverse transcriptase (Invitrogen)
`with oligo(dT)12–18 primer. PCR was performed using an Eppendorf Master-
`cycler gradient PCR system. The primers specific to rat MCT1 and MCT2
`were designed using Primer Express software and checked with the Blast
`analysis in the GenBank database for sequence identity (MCT1, forward
`5⬘-ACCCGAGACATCCGAAACC-3⬘
`and reverse 5⬘-AATTGTCCACT-
`GTCTGCACGG-3⬘; MCT2,
`5⬘-CCTCTTTGAATGTCTTATG-
`forward
`GACCA-3⬘
`5⬘-TATATCGAGCAATTTACCAGCCAG-3⬘;
`and
`reverse
`MCT3, forward 5⬘-GAGGATGTGGAGGCTGAGAG-3⬘ and reverse 5⬘-GC-
`CACCTATGGACTCGTGAT-3⬘; MCT4,
`forward 5⬘-GGGTCATCACTG-
`GCTTGGGT-3⬘ and reverse 5⬘-GGAACACGGGACTGCCTGC-3⬘; SMCT1,
`forward 5⬘-GTTGCTGGTGGGGATTCTTA-3⬘ and reverse 5⬘-CCACTGTG-
`GTCTGGGAAGTT-3⬘, and SMCT2, forward 5⬘-GAGAGCTTGTTGCTGT-
`TCCC-3⬘ and reverse 5⬘-GGAGTGGCTTAAGCTTGCAC-3⬘. The PCR reac-
`tion mixtures contained 200 ␮M concentrations of each dNTP, 0.1 ␮M
`concentrations of each forward and reverse primer, and 0.5 U/reaction Taq
`DNA polymerase in 1⫻ PCR buffer (500 mM KCl, 100 mM Tris-HCl, pH 8.3,
`and 15 mM MgCl2) through 35 cycles of 94°C for 0.5 min, 60°C for 0.5 min,
`
`Page 2 of 7
`
`

`
`1406
`
`CUI AND MORRIS
`
`and 72°C for 0.5 min. The PCR products were separated by electrophoresis on
`2% agarose gel, stained by ethidium bromide, and visualized under UV light.
`Data Analysis. The uptake data are presented as the mean ⫾ S.D. Data
`analysis was performed using GraphPad Prism (GraphPad Software Inc., San
`Diego, CA). Differences with p ⬍ 0.05 were considered statistically signifi-
`cant. The pH dependence of GHB uptake in the presence and absence of
`sodium was analyzed by a two-way ANOVA with a Bonferroni post-test. The
`uptake kinetic parameters, Michaelis-Menten constant (Km), and maximum
`velocity (Vmax), as well as the diffusional clearance (P), were determined by
`fitting the data using weighted nonlinear regression analysis (WinNonlin 5.0;
`Pharsight, Mountain View, CA) and eqs. 1 through 4:
`
`v ⫽
`
`Vmax 䡠 C
`Km ⫹ C
`
`v ⫽
`
`Vmax 䡠 C
`Km ⫹ C
`
`⫹ P 䡠 C
`
`v ⫽ P 䡠 C
`
`v ⫽
`
`Vmax1 䡠 C
`Km1 ⫹ C
`
`⫹
`
`Vmax2 䡠 C
`Km2 ⫹ C
`
`(1)
`
`(2)
`
`(3)
`
`(4)
`
`FIG. 1. mRNA expression of SMCT1, MCT1, and MCT2 in FRTL-5 cells. Studies
`were performed as described under Materials and Methods. bp, base pairs.
`
`where v is the uptake rate of GHB, C is the concentration of GHB, and P is the
`nonsaturable diffusion uptake clearance. The goodness of fit was determined
`by the sum of the squared derivatives, the residual plot, and the Akaike
`information criterion (AIC). The equation that provided the smallest coeffi-
`cient of variation percentage and AIC was used for obtaining Km, Vmax, and P
`parameters for the uptake data.
`The percentage of inhibition was calculated using eq. 5. The inhibition of
`GHB uptake by selected inhibitors (IC50) was calculated by fitting eq. 6 using
`weighted nonlinear regression analysis (WinNonlin 5.0):
`
`冊
`
`(5)
`
`(6)
`
`%Inhibition共F兲 ⫽ 100 ⫻冉 vNa⫹ ⫺ vinh
`
`vNa⫹ ⫺ vNa⫺
`
`F ⫽ 100 ⫻
`
`Imax ⫻ Cr
`r ⫹ Cr
`IC50
`
`⫹ is the uptake rate of GHB in the presence of sodium, vInh is the
`where vNa
`⫺ is
`uptake rate of GHB in the presence of both sodium and inhibitors, and vNa
`the uptake rate of GHB in the absence of sodium. F is the percentage of
`sodium-dependent uptake rate of GHB in the presence of inhibitors compared
`with the control and C is the concentration of inhibitors, Imax is the maximum
`percentage of inhibition, IC50 is concentration of inhibitor that provides 50%
`of maximal inhibition, and r is the Hill coefficient. The goodness of fit was
`determined by the sum of the squared derivatives, the residual plot, and the
`AIC.
`
`Results
`Gene Expression of SMCT and MCT Isoforms in FRTL-5
`Cells. The gene expression of SMCT1, SMCT2, and MCT1– 4 iso-
`forms in FRTL-5 cells was examined by RT-PCR with specific
`primers designed for each gene. Results showed that mRNAs of
`SMCT1, MCT1, and MCT2 were expressed in FRTL-5 cells (Fig. 1).
`mRNAs for SMCT2, MCT3, and MCT4 were not detected.
`Effect of Sodium on Cellular Uptake of D-Lactate, Butyrate,
`and GHB. To study the effect of sodium on the cellular uptake of
`known SMCT1 substrates D-lactate and butyrate, FRTL-5 cells
`were incubated with 100 nM [3H]D-lactate or 1 ␮M [14C]butyrate
`for 1, 5, and 30 min in the presence or absence of sodium at pH 7.4.
`Compared with the Na⫹-free condition, the uptake of D-lactate and
`butyrate in the presence of sodium is higher at 5 and 30 min for
`both compounds (Fig. 2A for D-lactate; Fig. 2B for butyrate).
`Likewise, uptake of 20 nM [3H]GHB was significantly higher in
`the presence of sodium than for the sodium-free condition at 5 and
`30 min.
`
`FIG. 2. Effect of sodium on the cellular uptake of D-lactate, butyrate, and GHB.
`FRTL-5 cells were incubated with 100 nM [3H]D-lactate (A), 1 ␮M [14C]butyrate
`(B), or 20 nM [3H]GHB (C) for 1, 5, and 30 min in the presence (䡺) or absence (f)
`of sodium at pH 7.4. Uptake values were normalized by protein concentration.
`Results are presented as the mean ⫾ S.D. The experiment was repeated three times
`with triplicate determinations in each experiment. Statistical analysis by a Student’s
`t test: ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01, compared with uptake in the absence of sodium.
`
`Time Course of GHB Cellular Uptake. FRTL-5 cells were incu-
`bated with 20 nM [3H]GHB for up to 60 min at room temperature. The
`uptake was linear up to 10 min (Fig. 3). Therefore, an incubation time
`of 5 min was chosen to determine uptake of GHB in all uptake studies.
`Effect of pH on GHB Uptake. The effect of extracellular pH on
`GHB uptake by FRTL-5 cells was examined by incubating cells with
`buffers of different pH (5.5, 6.0, 6.5, 7.0, and 7.4) containing 20 nM
`[3H]GHB for 5 min in the presence and absence of sodium (Fig. 4).
`The two-way ANOVA indicated that both sodium and pH affect
`uptake of GHB in the FRTL-5 cells with no significant interaction
`between the effects of sodium and pH. In the presence of sodium, the
`uptake rates at different pH values are not significantly different.
`However, in the absence of sodium, FRTL-5 cells exhibited increased
`
`Page 3 of 7
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`

`
`SODIUM-DEPENDENT MCT TRANSPORT OF GHB
`
`1407
`
`FIG. 3. Time course of GHB uptake in FRTL-5 cells. FRTL-5 cells were incubated
`with 20 nM [3H]GHB for up to 60 min in the presence (E) or absence (F) of sodium
`at pH 7.4. Uptake values were normalized by protein concentration. Results are
`presented as the mean ⫾ S.D. The experiment was repeated three times with
`triplicate determinations in each experiment. Statistical analysis by a Student’s t
`test: ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01, compared with uptake in the absence of sodium.
`
`FIG. 5. Concentration-dependent GHB uptake in FRTL-5 cells. Studies were per-
`formed at pH 7.4 with uptake determined at 5 min. A, the uptake data, obtained at
`various GHB concentrations, were best fitted to the eq. 2, as described under
`Materials and Methods. Data are expressed as the mean ⫾ S.D. of three experi-
`ments, with each experiment performed in triplicate. The symbols represent ob-
`served data and the lines represent the best fit of the data. B, Eadee-Hofstee plot of
`the data.
`
`were incubated with 10 ␮M [3H]GHB in the presence or absence of
`1 mM D-lactate, L-lactate, butyrate, pyruvate, BHB, ketoprofen, ibu-
`profen, naproxen, CHC, probenecid, or DIDS or with 0.05 mM
`luteolin or 0.25 mM phloretin. Uptake in the presence of sodium-
`containing buffer (without inhibitors) was used as the control. All
`uptake results are presented as percentage of control (Fig. 6A). GHB
`uptake was significantly inhibited by D-lactate, L-lactate, butyrate,
`pyruvate, BHB, ketoprofen, ibuprofen, naproxen, and probenecid.
`MCT inhibitors CHC, luteolin, and phloretin and the anion exchanger
`inhibitor DIDS had no inhibitory effect on GHB uptake in FRTL-5
`cells. Effects of ibuprofen, ketoprofen, and probenecid on the uptake
`of D-lactate and butyrate in FRTL-5 cells were also examined. Uptake
`of D-lactate was significantly inhibited by ketoprofen and ibuprofen
`(Fig. 6B). All three inhibitors significantly inhibited butyrate uptake
`(Fig. 6C).
`Concentration-dependent inhibition of GHB (10 ␮M) uptake was
`demonstrated for ibuprofen (Fig. 7A), ketoprofen (Fig. 7B), probene-
`cid (Fig. 7C), and L-lactate (Fig. 7D). FRTL-5 cells were incubated
`with 10 ␮M [3H]GHB in the presence of inhibitors for 5 min. The
`percent inhibition and IC50 values were calculated using eqs. 5 and 6.
`IC50 values were 31.6 ⫾ 19.7, 64.4 ⫾ 23.4, 380 ⫾ 43.4, and 101 ⫾
`37.3 ␮M (mean ⫾ S.D., n ⫽ 3) for ketoprofen, ibuprofen, probenecid,
`and L-lactate, respectively.
`Effect of Inhibitors on GHB Uptake in Rat MCT1 Gene-Trans-
`fected Cells. Expression of MCT1 protein and transport of GHB in
`MCT1 gene-transfected MDA-MB231 cells have been demonstrated
`previously (Wang et al., 2006). In this study, effects of SMCT1
`inhibitors on MCT1-mediated GHB (5 ␮M) uptake were examined for
`ibuprofen, ketoprofen, probenecid, and L-lactate. The uptake was
`determined by incubation of cells with [3H]GHB (5 ␮M) for 1 min at
`an external buffer pH of 6.0. Consistent with a previous report (Wang
`et al., 2006), GHB uptake was significantly increased in MCT1-
`transfected cells compared with mock-transfected cells, and the up-
`take was not affected by sodium. MCT1 inhibitors CHC (1 mM) and
`luteolin (50 ␮M) significantly reduced GHB uptake in MCT1-trans-
`fected cells. The four SMCT1 inhibitors ibuprofen, ketoprofen, pro-
`
`FIG. 4. Effect of pH on GHB uptake in FRTL-5 cells. Studies were performed using
`buffers at various pH values (pH 5.5, 6.0, 6.5, 7.0, and 7.4) with uptake determined
`at 5 min. In the absence of sodium, the cellular uptake at pH 6.5, 6.0, and 5.5 are
`significantly higher than that at pH 7.4 (p ⬍ 0.05). Each bar represents the mean ⫾
`S.D. of three experiments, each performed in triplicate. Statistical analysis by a
`two-way ANOVA: ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01, compared with uptake in the absence
`of sodium.
`
`GHB uptake when pH was decreased. The uptake rates at pH values
`less than 6.5 were significantly greater than that at pH 7.4. For all the
`pH values examined in this study, uptake in the presence of sodium is
`significantly higher than that for the sodium-free condition.
`The concentration dependence for GHB uptake was determined
`over a range of GHB concentrations (1 ␮M–10 mM) with uptake
`determined at 5 min and extracellular pH of 7.4. The uptake of GHB
`exhibited typical Michaelis-Menten kinetics in the presence of sodium
`and exhibited linear uptake in the absence of sodium (Fig. 5A). The
`uptake data obtained in the presence and absence of sodium were
`fitted to eqs. 1 through 4. We also determined the net uptake values
`for GHB in the presence of sodium by subtracting the values in the
`absence of sodium and fitted the net uptake data with eqs. 1 through
`4. The total uptake values were best fitted with eq. 2, and the net
`uptake data were best fitted with eq. 1, suggesting only one major
`transporter mediates GHB uptake in FRTL-5 cells in the presence of
`sodium (137 mM) at pH 7.4. The kinetic parameters (Km 0.68 ⫾ 0.30
`mM, Vmax 3.50 ⫾ 1.58 nmol 䡠 mg⫺1 䡠 min⫺1, and diffusional clear-
`ance of 0.25 ⫾ 0.08 ␮l 䡠 mg⫺1 䡠 min⫺1) were obtained by simulta-
`neously fitting the uptake data obtained in the presence and absence of
`sodium to eqs. 2 and 3. The data were also plotted using an Eadee-
`Hofstee plot (Fig. 5B). The linearity observed with this plot also
`suggested a single transporter-mediated process.
`Effect of Inhibitors on Sodium-Dependent GHB Uptake. The
`inhibitory effects of selected compounds on GHB uptake by FRTL-5
`cells were assessed at pH 7.4 in the presence of sodium. The cells
`
`Page 4 of 7
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`CUI AND MORRIS
`
`FIG. 7. Concentration-dependent inhibition of GHB uptake by ibuprofen (A), ke-
`toprofen (B), probenecid (C), and L-lactate (D). The symbols represent observed
`data, and the lines represent the fitted data. The uptake results were normalized by
`the control values (uptake in the presence of sodium and absence of inhibitors).
`Uptake in the absence of sodium has been subtracted from the values. The uptake
`studies were conducted at pH 7.4 and room temperature, and the uptake time was 5
`min. The concentration of GHB used was 10 ␮M. The experiments were repeated
`three times with triplicate determinations for each experiment. The data are pre-
`sented as the mean ⫾ S.D. for one representative experiment.
`
`FIG. 6. Effect of inhibitors on the uptake of GHB (A), D-lactate (B), and butyrate
`(C) by FRTL-5 cells. The uptake studies were conducted at pH 7.4 in the presence
`of sodium with an uptake time of 5 min. The concentration of GHB was 10 ␮M, and
`the concentration of all inhibitors was 1 mM, except for phloretin (0.25 mM) and
`luteolin (0.05 mM). The uptake values were normalized to that in the presence of
`sodium and without inhibitors. The data are presented as the mean ⫾ S.D. The
`experiments were repeated three times with triplicate determinations for each
`experiment. One-way ANOVA followed by Dunnett’s test was used for statistical
`analysis: ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01.
`
`benecid, and L-lactate also significantly decreased uptake of GHB in
`MCT1 gene-transfected cells (Fig. 8).
`Effect of Inhibitors on GHB Uptake at Different pH Values in
`FRTL-5 Cells. To investigate the transport mechanism of GHB by
`FRTL-5 cells, effect of inhibitors were examined at pH 6.0 and 7.4 for
`CHC, luteolin, ibuprofen, and L-lactate (Fig. 9). At pH 7.4, 90% of
`uptake was blocked in the absence of sodium compared with that
`under sodium-containing conditions, and the uptake was not further
`reduced by inhibitors. In the presence of sodium, uptake of GHB was
`significantly reduced by ibuprofen and L-lactate but not affected by
`CHC and luteolin. At pH 6.0, GHB uptake with the sodium-free buffer
`was reduced by 39%, on average, compared with that under sodium-
`containing conditions. Likewise, the uptake of GHB was significantly
`reduced by ibuprofen and L-lactate in the presence of sodium. In the
`absence of sodium, uptake was decreased to similar levels by all four
`inhibitors. However, only inhibition by CHC, luteolin, and ibuprofen
`was statistically significant.
`
`Discussion
`MCTs and SMCTs are the two plasma membrane transporter fam-
`ilies known to mediate the cellular uptake of monocarboxylic acids.
`MCTs belong to the solute carrier gene family SLC16A, and 14
`members of this family have been identified (MCT1–14) (Halestrap
`
`FIG. 8. Effect of SMCT1 inhibitors on GHB uptake in rat MCT1 gene-transfected
`MDA-MB231 cells. The uptake results were normalized by the control values
`(uptake by MCT1 gene-transfected cells in the presence of sodium and absence of
`inhibitors). The uptake studies were conducted at pH 6.0, and the uptake time was
`1 min. The concentration of GHB used was 5 ␮M, and the concentration of the
`inhibitors was 1 mM, except for luteolin (0.05 mM). The experiments were repeated
`three times, with triplicate determinations for each experiment. The data are pre-
`sented as the mean ⫾ S.D. for one representative experiment. Statistical analysis by
`Student’s t test: ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; compared with the control.
`
`and Price, 1999). MCTs mediate transport of monocarboxylates in a
`proton-dependent manner. SMCTs belong to the SLC5A family,
`containing two members (SMCT1 and SMCT2) with transport driven
`by a sodium gradient. Despite the differences in sequence identity and
`driving force, MCTs and SMCTs have similar substrate specificity
`(Srinivas et al., 2005). MCT-mediated GHB transport has been ex-
`tensively characterized using kidney cell lines and kidney membrane
`vesicles (Wang et al., 2006). In this study we found that GHB, as well
`as the monocarboxylates D-lactate and butyrate, undergo sodium-
`dependent uptake by FRTL-5 cells.
`
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`SODIUM-DEPENDENT MCT TRANSPORT OF GHB
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`2008) and is lower than that for rat MCT1-mediated GHB transport in
`gene-transfected cells (Km 4.6 mM) (Wang et al., 2006). For most of
`the common monocarboxylate substrates, SMCT1 possesses higher
`affinity than MCT1 (Halestrap and Price, 1999; Ganapathy et al.,
`2008).
`MCT and SMCT exhibit similar substrate specificity, necessitating
`the identification of specific inhibitors to discriminate the role of each
`transporter in drug disposition. Uptake of GHB at pH 7.4 in the
`presence of sodium was significantly inhibited by the substrates
`lactate, butyrate, pyruvate, and BHB but not by CHC, phloretin,
`luteolin, and DIDS. CHC is a known inhibitor of MCT, suggesting
`that MCTs do not play an important role in the uptake of GHB by
`FRTL-5 cells at pH 7.4. Luteolin and phloretin are naturally occurring
`flavonoids with potent inhibitory effects on GHB transport in rat
`MCT1 gene-transfected cells (Wang and Morris, 2007). In vivo stud-
`ies have demonstrated that luteolin and L-lactate can increase the renal
`clearance and total clearance of high-dose GHB in rats, suggesting a
`potential strategy for the treatment of GHB overdoses (Morris et al.,
`2005; Wang and Morris, 2007; Wang et al., 2008). The current study
`indicated that the underlying mechanism for the effect of luteolin and
`L-lactate may be different: a luteolin-GHB interaction is probably
`mediated by MCT, whereas L-lactate may inhibit both MCT and
`SMCT. Probenecid inhibits a wide range of efflux and influx trans-
`porters including MCTs, organic anion transporter, organic anion-
`transporting polypeptide, multidrug resistance-associated protein, and
`organic cation transporter (Shitara et al., 2005); in this study, probe-
`necid inhibited GHB transport by SMCT1 in FRTL-5 cells. Ibuprofen
`and other structurally related nonsteroidal anti-inflammatory drugs
`(NSAIDs) have been reported to inhibit uptake of nicotinate and
`propionate-invoked currents in mammalian cells or X. laevis oocytes
`expressing SMCT1. It was suggested that ibuprofen functions as an
`inhibitor but not substrate of SMCT1 (Itagaki et al., 2006). Our study
`demonstrated the inhibitory effect of ibuprofen and structurally re-
`lated NSAIDs on SMCT1-mediated uptake of D-lactate, butyrate, and
`GHB in FRTL-5 cells. The role of NSAIDs in MCT-mediated trans-
`port has not been described previously. To examine the specificity of
`SMCT1 inhibitors, we later tested their effects on GHB uptake in rat
`MCT1 gene-transfected MDA-MB231 cells. Both ibuprofen and ke-
`toprofen inhibited MCT1-mediated GHB uptake. Interactions of ibu-
`profen and ketoprofen with L-lactate and with benzoic acid have been
`observed in Caco-2 cells and Chinese hamster ovary cells, indicating
`that NSAIDs, L-lactate, and benzoic acid may represent substrates/
`inhibitors for a common transporter in these cells (Tamai et al., 1995;
`Choi et al., 2005). Our results demonstrated that ibuprofen and struc-
`turally related NSAIDs inhibited both SMCT1 and MCT1. The inhi-
`bition mechanism for each transporter requires further evaluation.
`Use of the rat thyroid cell line FRTL-5 in the present study allowed
`us to characterize the SMCT1-mediated transport of GHB and deter-
`mine substrates and inhibitors of this transporter. Species differences
`in substrate affinity have not been extensively studied; however,
`similar Km values for nicotinate have been reported for human,
`mouse, and rat SMCT1 (Paroder et al., 2006; Ganapathy et al., 2008).
`Results from studies using X. laevis oocyte