Drug Metabolism and Disposition Fast Forward
First published on May 14, 2007; DOI: 10.1124/dmd.107.014852
0090-9556/07/3508-1393-1399$20.00
DMD 35:1393-1399, 2007
The Role of Monocarboxylate Transporter 2 and 4 in the Transport of 
-Hydroxybutyric Acid in Mammalian Cells
Qi Wang, and
Marilyn E. Morris
Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, New York
(Received January 19, 2007;
accepted May 10, 2007)
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Abstract
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Monocarboxylate transporter 1 (MCT1) is an important determinant of the renal transport of the drug of abuse, 
-hydroxybutyric acid (GHB). The objective of this study was to investigate the role of MCT2 and MCT4, present in tissues including intestine, kidney, skeletal muscle, and brain, in the membrane transport of GHB and the MCT substrate L-lactate. mRNA and protein of MCT2 and MCT4 were expressed in MDA-MB231 cells, as detected by reverse transcription-polymerase chain reaction and Western blot analysis; MCT1 and MCT3 were not detected. The uptake of GHB or L-lactate by MDA-MB231 cells was pH-dependent but not sodium-dependent. The concentration-dependent uptake of GHB was best fitted to a single-transporter model with a diffusional clearance component (Km of 17.6 ± 1.5 mM, Vmax of 50.6 ± 9.0 nmol · mg–1 min–1 and diffusional clearance of 0.20 ± 0.07 µl · mg–1 min–1). On the other hand, the concentration-dependent uptake of L-lactate was best fitted to a two-transporter model (Km of 21 ± 2.5 and 3.0 ± 1.5 mM, and Vmax of 268 ± 72 and 62.9 ± 42.2 nmol · mg–1min–1, respectively). The uptake of GHB and L-lactate was inhibited by MCT inhibitors 
-cyano-4-hydroxycinnamate (CHC), phloretin, and p-chloromercuribenzoic acid; CHC inhibited GHB and L-lactate uptake with IC50 values of 1.71 ± 0.39 and 0.71 ± 0.11 mM, respectively. Small interfering RNA treatment to silence MCT2 or MCT4 significantly decreased their protein expression and the uptake of L-lactate and GHB; however, the decrease in GHB uptake with MCT2 inhibition was smaller than that for MCT4. This investigation demonstrated that GHB is a substrate for both MCT2 and MCT4; these transporters may be important in the nonlinear disposition of GHB, as well as influencing its tissue distribution.
-Hydroxybutyric acid (GHB) is present in mammalian brain as a metabolite of -aminobutyric acid (Bessman and Fishbein, 1963
); studies have revealed that GHB is a neurotransmitter or neuromodulator with its own specific receptors (Snead, 2000). GHB has been detected in various tissues other than brain, including heart, kidney, liver, lung, muscle, and gastrointestinal tract (Nelson et al., 1981; Tedeschi et al., 2003). GHB is approved in the United States for treatment of the sleep disorder narcolepsy (Mamelak et al., 1986) and is used in the treatment of alcohol dependence in Europe (Gallimberti et al., 2000). The abuse of GHB by body builders as a popular steroid alternative, due to its growth hormone-releasing effects (Okun et al., 2001), and by drug abusers as a recreational drug at nightclubs and rave parties for its euphoric effects, has resulted in serious adverse effects, including coma, seizure, and death (Mason and Kerns, 2002).
The pharmacokinetics of GHB has been reported to be nonlinear in rats (Lettieri and Fung, 1979) and humans (Palatini et al., 1993; Scharf et al., 1998). The nonlinear pharmacokinetics of GHB is due to capacity-limited metabolism (Lettieri and Fung, 1979; Palatini et al., 1993), absorption (Arena and Fung, 1980), and renal elimination (Morris et al., 2005). The distribution of GHB to various tissues, including the brain, is also dependent on specific transporters since, at physiologic pH, more than 99% of GHB is ionized and cannot readily diffuse across cellular membranes.
Our recent studies have demonstrated that the monocarboxylate transporter 1 (MCT1) is important in the membrane transport of GHB (Morris et al., 2005; Wang et al., 2006a). Monocarboxylate transporters belong to the SLC16A gene family and consist of 14 members that differ in terms of substrate specificities and affinities (Halestrap and Meredith, 2004). MCTs 1 to 4 have been demonstrated to be proton-coupled transporters (Halestrap and Meredith, 2004) with similar, but not identical, substrates, including the endogenous compounds, lactate and pyruvate (Garcia et al., 1994; Lin et al., 1998) and the exogenous compounds, foscarnet, nateglinide, simvastatin acid, lovastatin acid, and fluorescein (Tsuji A, 1993; Nagasawa et al., 2002; Okamura et al., 2002). However, the tissue distribution of MCT isoforms is quite different. MCT1 is ubiquitously distributed, being present in the intestine, colon, muscle, heart, brain, kidney, and red blood cells (Garcia et al., 1994; Lin et al., 1998; Price et al., 1998). The tissue distribution of MCT2 is more restricted, with MCT2 being found mainly in testis, brain, skeletal muscle, heart, and kidney (Lin et al., 1998). MCT4 is present in testis, small intestine, lung, brain, heart, kidney, and spleen (Dimmer et al., 2000). Our previous studies with rat kidney cortex membrane vesicles suggested that MCT1 mediated, at least in part, the transport of GHB in mammalian kidney (Wang et al., 2006a); however, it is likely that MCT2 and MCT4, which have been shown to transport L-lactate, may also play a role in the membrane transport and disposition of GHB.
The objectives of this study were 1) to characterize the mRNA and protein expression of MCT1, MCT2, MCT3, and MCT4 in MDA-MB231 cells; 2) to investigate the transport driving forces and transport kinetics of GHB and the MCT substrate L-lactate; 3) to study the effect of MCT inhibitors on the uptake of GHB; and 4) to investigate the role of MCT2 and MCT4 in GHB transport through the use of RNA interference studies.
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Materials and Methods
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L-Lactate, -cyano-4-hydroxycinnamate (CHC), p-chloromercuribenzoic acid (pCMB), phloretin, and tetraethylammonium chloride (TEA) were purchased from Sigma (St. Louis, MO). [2,3-3H]-Hydroxybutyric acid (50 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA). L-[U-14C]Lactate (56 mCi/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). MDA-MB231 cells were purchased from The American Type Culture Collection (Manassas, VA).
Cell Culture. MDA-MB231 cells were maintained in RPMI 1640 (Invitrogen Corp., Carlsbad, CA) with 5% fetal bovine serum, 100 units of penicillin, and 100 µg/ml streptomycin, at 37°C in a 5% CO2/95% air environment. Cells reached confluence after 5 to 7 days in culture. Cells were subcultured at a ratio of 1:4 using 0.05% trypsin and 0.53 mM EDTA, and were seeded in 35-mm (diameter) plastic culture dishes 2 days before uptake studies.
RT-PCR. The primers specific to human MCT1, MCT2, MCT3, and MCT4 have been used in other studies to detect mRNA in our laboratory (Wang et al., 2006b). In brief, first-strand cDNA was synthesized from the total RNA isolated from MDA-MB231 cells. The PCRs were performed with 200 µM concentrations of each deoxynucleoside-5'-triphosphate, a 0.2 µM concentration of each forward and reverse primer, and 0.5 U/reaction Taq DNA polymerase in 1x PCR buffer through 40 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min. The PCR products were separated by electrophoresis on 2% agarose gel, and then stained by ethidium bromide and visualized under UV light. The PCR products were also purified using QIAquick PCR clean-up kit (QIAGEN Inc. Valencia, CA) and then sequenced.
Western Blot Analysis. The expression of MCT1, MCT2, and MCT4 in MDA-MB231 cells was examined by Western blot. MDA-MB231 cells were harvested and lysed with lysis buffer [0.15 M NaCl, 5 mM EDTA, 1% Triton X-100, 10 mM Tris-Cl pH 7.4, 5 mM dithiothreitol, 100 µM phenylmethane-sulfonyl fluoride in isopropanol, 5 mM 
-aminocaproic acid] on ice for 30 min and then centrifuged at 16,000g for 18 min at 4°C. The supernatants were collected and separated on a 10% (w/v) polyacrylamide gel containing 0.1% (w/v) SDS and then transferred to a nitrocellulose membrane. After blocking with 5% (w/v) milk (Bio-Rad, Hercules, CA) in Tris-buffered saline containing 0.05% (v/v) Tween 20 at 20°C for 1 h, the membranes were incubated with primary antibodies for MCT1 (1 µg/ml), MCT2 (1 µg/ml), and MCT4 (1 µg/ml) (U. S. Biological, Swampscott, MA) overnight at 4°C. After incubation with specific horseradish peroxidase-conjugated secondary antibodies (Chemicon, Temecula, CA) for 1 h at room temperature, the membranes were developed with the ECL system (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK).
Uptake Studies. Growth medium was removed from the cell monolayers and cells were washed three times with uptake buffer (150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.5). Uptake was initiated by adding 1 ml of buffer containing 0.05 µCi of L-[14C]lactate or 0.5 µCi of [3H]GHB to the dishes. To investigate the effect of pH on uptake, the cells were incubated with uptake buffer with different pH values (pH 6.0 and 7.5). To investigate the effect of Na+ on uptake, the cells were incubated with uptake buffer in the presence of Na+ (150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.5, or 10 mM 2-(N-morpholino)ethane-sulfonic acid, pH 6.0) or in the absence of Na+ (150 mM N-methyl-D-glucamine, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.5, or 10 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.0). To investigate the time dependence of the uptake, cells were incubated at room temperature for 0.5, 1, 5, 10, 30, 60, and 90 min (if incubated longer than 90 min, the cells began to detach from the dishes). From these studies, a time point of 10 min was chosen to represent the linear uptake of GHB and 1 min to represent the linear uptake of L-lactate. A short uptake period for L-lactate was chosen to minimize the cellular metabolism of L-lactate; no metabolism of GHB occurs over this 10-min period as determined in a preliminary study. Several inhibitors were used in cellular uptake studies including CHC, pCMB, TEA, and phloretin. The uptake was stopped by aspirating the buffer, and the cells were immediately washed three times with ice-cold stop buffer (150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.5). The cells were lysed with 1 ml of lysis buffer containing 0.3 N NaOH and 1% SDS, and the radioactivity was determined using a liquid scintillation counter (1900 CA Tri-carb liquid scintillation analyzer; PerkinElmer Life and Analytical Sciences, Boston, MA). Protein concentrations were determined using the BCA protein assay kit (Pierce Chemicals, Rockford, IL) with bovine serum albumin as standard. The results were normalized for the protein content of the cells in each dish and accumulation was expressed as pmol or nmol/mg protein/min.
RNA Interference. Two siRNA constructs targeting MCT1, two siRNA constructs targeting MCT2 (exon 4 and exon 5, respectively) and two siRNA constructs targeting MCT4 (exon 5), were designed using the manufacturer's supplied tools and purchased from Ambion Inc. (Austin, TX). The sequences of siRNA for MCT1 are (sense/antisense, 5'-3'): #1, GCAGUAUCCUGGUGAAUAAtt, UUAUUCACCAGGAUACUGCtg. The sequences of siRNA for MCT2 are: #5, CCCUUGAGCAAAUCUAAACtt, GUUUAGAUUUGCUCAAGGtt and #6, CCCGCCUUAACCAUAAUUGtt, CAAUUAUGGUUAAGGCGGGtt. The sequences of siRNA for MCT4 are: #3 CGUCUACAUGUACGUGUUCtt, GAACACGUACAUGUAGACGtg and #4, CCCACGUCUACAUGUACGUtt, ACGUACAUGUAGACGUGGGtc. The nonspecific scrambled control RNA was also purchased from Ambion Inc. and was used as a negative control. For the transfection experiments, cells were seeded in six-well plates at 20 to 40% confluence 1 day before transfection. Cells were transfected with siRNA at a final concentration of 20 nM using Lipofectamine 2000 (Invitrogen Corp.), according to the manufacturer's instructions. The cells were then incubated at 37°C in 5% CO2 for 48 to 96 h before the Western blot analysis and uptake experiments.
Data Analysis. All the uptake data are presented as mean ± S.D. One-way analysis of variance (ANOVA) followed by Dunnett's test was used to detect statistical significance among means of more than two groups. The differences with a P value of 0.05 or less were considered as statistically significant. Data analysis was performed using GraphPad Prism (GraphPad Inc., San Diego, CA). The transport kinetic parameters Michaelis-Menten constant, Km, and maximum uptake rate, Vmax, were determined by fitting the data using weighted nonlinear regression analysis [Adapt II; Biomedical Simulations Resource (BMSR), University of South California, Los Angels, CA] using the following equations:
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Here v is the uptake rate of L-lactate or GHB and C is the concentration of L-lactate or GHB. P is the nonsaturable uptake clearance. The symbol I represents the inhibitor concentration. Ki is the inhibition constant: Ki_low is the inhibition constant for low-affinity transporter, and Ki_high is the inhibition constant for high-affinity transporter. The goodness of fit was determined by the sum of the squared derivatives, the residual plot, and the Akaike information criterion (Kamikubo et al., 1986). The equation that provided the smallest CV% and Akaike information criterion for the data was used in obtaining Km and Vmax parameters for the uptake data.
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FIG. 1. Expression of MCT isoforms in MDA-MB231 cells. A, the RT-PCR product of MCT2 (251 base pairs) and MCT4 (200 base pairs). B, protein expression of MCT2 (36 kDa) and MCT4 (50 kDa). The Western blots were treated with MCT2 or MCT4 antibodies first, and then treated with the ß-actin antibody. Upper lanes show MCT isoform protein; lower lanes show ß-actin as the loading control.
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Results
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RT-PCR and Western Blot Results. Using RT-PCR, we demonstrated that mRNA expression for MCT2 and MCT4 was present in MDA-MB231 cells (the PCR products were confirmed by sequencing) (Fig. 1A), whereas MCT1 and MCT3 were not detected. MCT2 and MCT4 protein were also present in MDA-MB231 cells (Fig. 1B).
Uptake Studies. Sodium and pH effects on L-lactate uptake. A 1-min uptake time was used to study the L-lactate uptake by MDA-MB231 cells to minimize the influence of L-lactate metabolism. Uptake of L-lactate by MDA-MB231 cells was pH-dependent, with a significantly higher uptake rate at pH 6.0 than that at pH 7.5 (Fig. 2A). At pH 6.0, the uptake of L-lactate was not affected by the presence or absence of sodium, whereas at pH 7.5, a significant reduction was observed in the absence of sodium (Fig. 2A). The uptake of L-lactate could be inhibited to the same level when using the classical MCT inhibitor CHC at the two pH values (Fig. 2A).
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FIG. 2. Uptake of L-lactate in MDA-MB231 cells: driving forces and concentration dependence. A, pH and sodium effects on the uptake of L-lactate (0.5 µM). The sodium buffer contained 150 mM sodium, whereas the non-sodium buffer substituted N-methyl-D-glucamine for the sodium. All the uptake data were normalized to the uptake at pH 7.5 in the presence of sodium and compared with this uptake. B, concentration-dependent uptake at pH 6.0, with the line representing the model fitting of the data. C, Eadie-Hofstee plot of the concentration-dependent data. Data are presented as mean ± S.D., n = 3 to 4 independent experiments with triplicate determinations. One-way ANOVA followed by a Dunnett's test was used to detect statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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Kinetics of L-lactate uptake. Concentration dependence of L-lactate uptake at pH 6.0 was demonstrated in this study, and the uptake rate was fitted by eqs. 1, 2, and 3. An Eadie-Hofstee plot indicated that more than one transporter might be involved in the uptake of L-lactate (Fig. 2C). A two-transporter model described the data well with fitted Km_low and Km_high for low- and high-affinity transporters of 21.3 mM and 3.0 mM, and Vmax_low and Vmax_high for low- and high-affinity transporters of 268 and 62.9 nmol · mg–1 min–1, respectively (Table 1; Fig. 2B).
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TABLE 1 Kinetic parameters for the uptake of L-lactate and GHB
The parameters are presented as mean ± S.D., n = 3 to 4 independent experiments with triplicate determinations.
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Effects of MCT inhibitors on L-lactate uptake. The uptake of L-lactate by MDA-MB231 cells was significantly inhibited by the MCT inhibitors, phloretin (0.1 mM), pCMB (0.1 mM), and CHC (2 mM) to approximately 20% of control (Fig. 3A). A concentration-dependent inhibition of L-lactate uptake by CHC was demonstrated (Fig. 3B), and the IC50 value was determined to be 0.71 mM (Table 1). CHC can competitively inhibit L-lactate uptake in human kidney HK-2 cells that express predominantly MCT1 (Wang et al., 2007), and has been reported to be a competitive inhibitor for MCTs (Halestrap and Price, 1999). Therefore, competitive inhibition models were used to obtain the inhibition constants with the two-transporter model. The inhibition constants were determined to be 2.1 mM, and 0.03 mM for high and low Km transporters, respectively.
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FIG. 3. Uptake of L-lactate in MDA-MB231 cells: effects of inhibitors. A, effects of selected compounds on the uptake of L-lactate. The concentration of L-lactate was 0.5 mM, and those for CHC, phloretin, pCMB, and TEA were 2, 0.1, 0.1, and 5 mM, respectively. B, concentration-dependent inhibition of L-lactate (0.5 mM) uptake by CHC, with the line representing the model-fitting of the data to a competitive inhibition model. Data are presented as mean ± S.D., n = 3 to 4 independent experiments with triplicate determinations for each experiment. One-way ANOVA followed by a Dunnett's test were used to detect statistical difference. **, P < 0.01.
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Sodium and pH effects on GHB uptake. Uptake of GHB by MDA-MB231 cells was pH-dependent, with a significantly higher uptake rate at pH 6.0 than at pH 7.5 (Fig. 4A). At pH 6.0, the uptake of GHB was not affected by the presence or absence of sodium, whereas at pH 7.5, a significant reduction was observed in the absence of sodium (P < 0.05) (Fig. 4A). The uptake of GHB was inhibited to similar levels (no statistical difference) when using the classical MCT inhibitor CHC at the two pH values of 6.0 and 7.4 (Fig. 4A).
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FIG. 4. Uptake of GHB in MDA-MB231 cells: driving forces and concentration dependence. A, pH and sodium effects on the uptake of GHB (5.7 nM). The sodium buffer contained 150 mM sodium, whereas the non-sodium buffer substituted N-methyl-D-glucamine for the sodium. All the uptake data were normalized to the uptake at pH 7.5 in the presence of sodium. B, concentration-dependent uptake at pH 6.0, with the lines representing the model-fitting of the data. C, Eadie-Hofstee plot the of concentration-dependent data. Data are presented as mean ± S.D., n = 3 to 4 independent experiments with triplicate determinations for each experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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Kinetics of GHB uptake. The concentration-dependent uptake of GHB at pH 6.0 was demonstrated in this study (Fig. 4B). An Eadie-Hofstee plot suggested that more than one transporter or a transporter plus a non-transporter-mediated process might be involved in the uptake of L-lactate (Fig. 4C). A model with one transporter plus a diffusional component described the data well with fitted Km and Vmax of 17.6 mM and 50.6 nmol · mg–1 min–1, respectively, and a diffusional clearance of 0.20 µl · mg–1 min–1 (Table 1; Fig. 4B).
Effects of MCT inhibitors on GHB uptake. The uptake of GHB by MDA-MB231 cells was significantly inhibited by the MCT inhibitors, phloretin (0.1 mM), pCMB (0.1 mM), and CHC (2 mM) to approximately 60% of control, but not by an organic cation transporter inhibitor, TEA (Fig. 5A). A concentration-dependent inhibition of GHB uptake by CHC was demonstrated (Fig. 5B), and the IC50 value was determined to be 1.71 mM (Table 1). The uptake of GHB in the presence of 10 mM CHC was approximately 30% of control, which also suggested a non-transporter-mediated uptake of GHB by MDA-MB231 cells. Since previous studies have indicated that CHC is a competitive inhibitor for MCT-mediated GHB transport (Wang et al., 2007), we used eq. 5 to obtain an estimate of the inhibition constant. For these inhibition data, we also examined other kinetic models as described by eqs. 4 and 6; however, evaluation of goodness of fit indicated that eq. 5 provided the best fit of the data. The fitted Ki value was 1.8 mM, which is close to the Ki_low (2.1 mM) for the CHC inhibition of L-lactate.
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FIG. 5. Uptake of GHB in MDA-MB231 cells: effects of inhibitors. A, effects of selected inhibitors on the uptake of GHB in MDA-MB231 cells. GHB concentration was 0.1 mM, and the concentrations of CHC, phloretin, pCMB, and TEA were 2, 0.1, 0.1, and 5 mM, respectively. B, concentration-dependent uptake of GHB in MDA-MB231 cells at pH 6.0. The line represents the model-fitting of the data to a competitive inhibition model. Data are presented as mean ± S.D., n = 3to4 independent experiments with triplicate determinations for each experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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Effects of siRNA Treatment in MDA-MB231 Cells. Treatment with siRNAs for MCT4. Transfection of MDA-MB231 cells with either of the two siRNAs for MCT4 significantly decreased the protein expression of MCT4 in MDA-MB231 cells as determined by Western blot analysis (Fig. 6A). These two siRNAs for MCT4 had no effect on the protein expression of MCT2 (data not shown). The siRNA for MCT1 had no effect on the protein expression of MCT4 or MCT2, and the uptake of GHB or L-lactate by MDA-MB231 cells was not affected by the siRNA treatment for MCT1 (data not shown). Using 14C-L-lactate as the substrate, the uptake was significantly decreased after siRNA treatment for MCT4, with number 4 siRNA being the most effective (Fig. 6B). The uptake of GHB was significantly decreased to 60% of the negative control in cells after siRNA treatment for MCT4 (Fig. 6C).
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FIG. 6. Effects of treatment with MCT4 siRNA in MDA-MB231 cells. A, MCT4 protein expression after treatment of MDA-MB231 cells with two siRNAs for MCT4, with ß-actin used as the loading control. B, uptake of L-lactate (0.5 µM) in siRNA-treated MDA-MB231 cells. C, uptake of GHB (0.1 mM) in siRNA-treated MDA-MB231 cells. #3 and #4 are the two siRNAs for MCT4 used in the study (see Materials and Methods for details). Data are presented as mean ± S.D., n = 6to9. **, P < 0.01.
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Treatment with siRNAs for MCT2. Transfection of MDA-MB231 cells with either of the two siRNAs for MCT2 significantly decreased the protein expression of MCT2 (Fig. 7A). These two siRNAs had no effect on the protein expression of MCT4 as examined by Western blot analysis (data not shown). The uptake of L-lactate was significantly inhibited by one of the siRNA treatments (number 6), although the inhibition was moderate, with only a 17% decrease in uptake (Fig. 7B). The uptake of GHB after siRNA treatment (with treatment 6) was significantly lower than that of the negative control (Fig. 7C); as observed with L-lactate, the inhibition was modest, with only 15% inhibition (Fig. 7C).
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FIG. 7. Effects of treatment with MCT2 siRNA in MDA-MB231 cells. A, MCT2 protein expression after treatment of MDA-MB231 cells with two siRNAs for MCT2, with ß-actin used as the loading control. B, uptake of L-lactate (0.5 µM) in siRNA-treated MDA-MB231 cells. C, uptake of GHB (0.1 mM) in siRNA-treated MDA-MB231 cells. #5 and #6 are the two siRNAs for MCT2 used in the study (see Materials and Methods for details). Data presented as mean ± S.D., n = 6to9. *, P < 0.05; **, P < 0.01; ***P < 0.001.
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Discussion
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GHB is a monocarboxylic acid with a pKa value of 4.7, which is more than 99% ionized at physiological pH. The transport of GHB across various biological barriers and cellular membranes, such as blood-brain barrier, brain cells, and proximal tubule cells, requires specific transporters (Benavides et al., 1982; Bhattacharya and Boje, 2004; Wang et al., 2006a). The renal transport of GHB has been studied previously, and MCT1 represents an important transporter for GHB transport across the proximal tubule cells (Wang et al., 2006a). Pyruvate uptake by MCT2-transfected oocytes can be inhibited by GHB, suggesting that GHB may be a substrate of MCT2 (Lin et al., 1998).
L-Lactate, a known substrate of MCT2 and MCT4 (Halestrap and Meredith, 2004), exhibited decreased uptake in this investigation in MDA-MB231 cells after siRNA treatment for either MCT2 or MCT4. Kinetic studies of L-lactate demonstrated a high-affinity transporter with a Km of 3 mM and a low-affinity transporter with a Km of 21 mM; these values are similar to the previously reported values for MCT2 (Garcia et al., 1995; Lin et al., 1998)- and MCT4-mediated (Dimmer et al., 2000; Manning Fox et al., 2000) transport of L-lactate, respectively. The Ki values for CHC for the low-affinity and high-affinity transporters were also similar to previously reported Ki values for MCT2 and MCT4, respectively (Juel, 1997; Broer et al., 1999). The transport of GHB by MCT2 or MCT4 has not been previously studied. Kinetic data of GHB transport in MDA-MB231 cells supported a model with a single transporter plus a diffusional component; however, RNA interference studies for both MCT2 and MCT4 demonstrated significant reductions in the uptake of GHB. The activity of the high-affinity transporter, possibly MCT2, could not be identified using model fitting techniques, even in the presence of a transport inhibitor. The Ki value (1.8 mM) determined for GHB inhibition may well represent the Ki for the low-affinity transporter, which is very similar to that for the low-affinity L-lactate transporter (2.1 mM). Overall, our data suggested that both MCT2 and MCT4 represent transporters of GHB, but the contribution of MCT2 to GHB uptake in MDA-MB231 cells was small. Indeed, the inhibition of GHB uptake after MCT4 siRNA treatment was able to decrease the uptake to an extent similar to that of the chemical inhibitors CHC and phoretin; on the other hand, the inhibition of GHB uptake after MCT2 siRNA treatment only explained 15% of the total GHB uptake, indicating that it is a relatively minor component of the overall uptake. The transport of GHB by MDA-MB231 cells with a Km value of 17.6 mM was much lower than a previous estimation using an indirect fluorescence indicator pHi method (> 500 mM) (Manning Fox et al., 2000). This difference in Km value may be explained partly by the experimental differences in the studies, including the buffer pH used. It has been demonstrated previously that the affinity of substrates to MCT increases as pH decreases (Halestrap and Price, 1999).
The transport of L-lactate or GHB at pH 7.5 was significantly affected by the presence or absence of sodium, which suggested that a sodium-dependent transporter(s) might be involved in L-lactate or GHB transport. This sodium effect was not observed at pH 6.0, suggesting that with a low pH as the driving force, MCT transporters represent the predominant transporters for L-lactate and GHB.
GHB has been found in several extraneuronal tissues, including gastrointestinal tract, kidney, muscle, heart, liver, lung, brain fat, and blood (Nelson et al., 1981; Tedeschi et al., 2003). The endogenous concentrations of GHB in the kidney, gastrointestinal tract, heart, and muscle are much higher than those in brain and blood; however, the origin and function of GHB in extraneuronal tissues are not known. MCT1, MCT2, and MCT4 have been found to be present in brain, muscles, kidney, heart, gastrointestinal tract, and blood cells (Pellerin et al., 1998; Eladari et al., 1999; Bergersen et al., 2002; Merezhinskaya et al., 2004; Gill et al., 2005). The presence of these MCT transporters in these cellular or subcellular membranes enables the transport of GHB among various tissue types; therefore, monocarboxylate transporters are important in the physiological/pharmacological action of GHB, as well as in the absorption and disposition of GHB in the body.
It has been reported that absorption of GHB from the gastrointestinal tract was capacity-limited (Arena and Fung, 1980); however, the identity of possible transporters was not investigated. MCT1, MCT2, and MCT4 have been reported to be present in the gastrointestinal tract, with MCT1 exhibiting an apical membrane localization and MCT4 localized to the basolateral membrane (Gill et al., 2005). The renal reabsorption of GHB is also capacity-limited and represents a major mechanism of elimination after GHB overdoses (Morris et al., 2005). We have demonstrated previously that MCT1 mediated the transport of GHB in rat kidney cortex; however, whether MCT2 also represents a GHB transporter was unclear (Wang et al., 2006a). In this study, we demonstrate that MCT2 is indeed a GHB transporter. MCT2 has been demonstrated to be present in the kidney in the medullary thick limbs of Henle where MCT1 is not present (Eladari et al., 1999), so that it may represent an important GHB transporter at the medullary region of kidney. The transport of GHB across the blood-brain barrier (BBB) may also be transporter-mediated, and an MCT isoform was proposed as an uptake transporter (Bhattacharya and Boje, 2004). MCT1 has been shown to be present on both the abluminal and luminal membranes of BBB endothelial cells (Gerhart et al., 1997), suggesting that MCT1 may represent a major transporter for GHB at the BBB. MCT4 has been reported to be strongly expressed in astrocytes (Bergersen et al., 2002; Pellerin et al., 2005), suggesting the MCT4-mediated transport of GHB from BBB to neurons. MCT2 is a major MCT transporter in neurons and is present in the postsynaptic membrane of parallel fiber-Purkinje cell synapses (Bergersen et al., 2002, 2005), suggesting its role in the uptake of GHB into neurons. More studies are needed to investigate the role of MCT isoforms in GHB transport in the central nervous system.
In summary, this investigation using MDA-MB231 cells that express MCT2 and MCT4 has demonstrated that the transport of the monocarboxylates L-lactate and GHB are mediated by both MCT2 and MCT4. MCT4 represents the major transporter for GHB in these cells, with MCT2 responsible for a minor component of the uptake of GHB. These transporters may be important in both the bioavailability and renal clearance of GHB, as well as influencing its pharmacological activity by influencing its brain uptake and distribution.
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Footnotes
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Financial support was provided by National Institutes of Health Grant DA14988 and a grant from the Western New York Kidney Foundation/Upstate NY Transplantation Service.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.107.014852.
ABBREVIATIONS: GHB, -hydroxybutyrate; MCT, monocarboxylate transporter; CHC, -cyano-4-hydroxycinnamate; pCMB, p-chloromercuribenzoic acid; TEA, tetraethylammonium chloride; RT-PCR, reverse transcription-polymerase chain reaction; siRNA, small interfering RNA; ANOVA, analysis of variance; BBB, blood-brain barrier.
Address correspondence to: Dr. Marilyn E. Morris, 517 Hochstetter Hall, Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst, NY 14260. E-mail: memorris{at}buffalo.edu
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Abstract
Materials and Methods
