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Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, Korea (H.-J.K., S.-S.L., C.-H.L., J.-C.S., H.J.S., K.-H.L., J.-G.S.); and Department of Molecular Biology, College of Natural Sciences, Busan National University, Busan, Korea (H.-J.K., M.-A.Y.)
(Received December 26, 2005; accepted April 14, 2006)
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Abstract |
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; Igarashi et al., 1995). Haloperidol pyridinium metabolites are structurally similar to the neurotoxin MPP+ (1-methyl-4-phenylpyridinium; Fig. 1), which is known to cause neurodegeneration, and have been reported as having similar or more potent neurotoxic effects, e.g., inhibition of mitochondrial respiration, irreversible inhibition of tyrosine hydroxylase, and depletion of dopamine and serotonin (Bloomquist et al., 1994; Rollema et al., 1994; Fang et al., 1995). The formation of toxic pyridinium metabolites of haloperidol is mediated primarily by hepatic cytochrome P450 3A (Igarashi et al., 1995), although it has not been clearly established how the cationic metabolite HPP+ formed in peripheral tissues becomes localized to the brain (Igarashi and Castagnoli, 1992; Igarashi, 1998).
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). Furthermore, HPP+ shows a moderate brain uptake index, which indicates that it may permeate the blood-brain barrier. These results support the hypothesis that HPP+ is formed mainly in the liver, circulates in the bloodstream, and enters the brain, where it induces damage to brain dopaminergic neurons (Kawashima et al., 2002, 2004).
Recent reports have suggested that MPP+ is a high-affinity substrate for the human organic cation transporters hOCT1, hOCT2, and hOCT3 (Hayer-Zillgen et al., 2002). The hOCT1 and hOCT2 forms are expressed predominantly in the liver and kidneys, respectively (Gorboulev et al., 1997), whereas hOCT3 (also known as extraneuronal monoamine transporter), which also has a high affinity for monoamines, is highly expressed in the placenta (Verhaagh et al., 1999). Recently, it has been reported that hOCT3 may play a significant role in the disposition of cationic neurotoxins and neurotransmitters in the brain (Wu et al., 1998). Because MPP+ is metabolically stable as a charged molecule and shows low-level diffusion across the cytoplasmic membrane, it accumulates exclusively in nerve cells, in a distribution process that may be mediated by hOCTs (Russ et al., 1992). Although HPP+ is less potent than MPP+ in the dopaminergic system, these compounds display comparable toxic effects on the serotonergic system. Considering the structural similarities between MPP+ and both haloperidol metabolites, it seems likely that these metabolites represent substrates for hOCTs.
In the present study, we evaluated whether the toxic haloperidol pyridinium metabolites HPP+ and RHPP+ are substrates for hOCTs. In addition, we characterized HPP+ and RHPP+ uptake into hOCT-overexpressing MDCK cells to clarify the potential contribution of hOCTs to the disposition of these two metabolites.
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Materials and Methods |
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Cell Culture and Stable Transfection of hOCT1, hOCT2, and hOCT3. Caco-2 cells were maintained in a humidified atmosphere of 5% CO2 in air and were grown in DMEM that was supplemented with 20% FBS, 2 mM L-glutamine, 1% nonessential amino acids, and 100 U/ml penicillin-streptomycin. The medium was changed every second day. For the uptake assay, 5 x 105 Caco-2 cells were seeded on 12-well plastic cell culture clusters. The uptake studies were usually performed 7 to 9 days after the cells formed a monolayer. MDCK cells were maintained in a humidified atmosphere of 5% CO2 in air and were grown in DMEM that was supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/ml penicillin-streptomycin. For the construction of stable hOCT1-, hOCT2-, and hOCT3-expressing cells, the MDCK cells were transfected with pcDNA3.1 plasmids that carried the genes for hOCT1, hOCT2, and hOCT3, respectively, using Lipofectamine 2000. The stable cells were selected for resistance to 800 mg/ml geneticin (G418; Life Technologies, Karlsruhe, Germany) for 3 weeks (Hayer-Zillgen et al., 2002). The expression levels of hOCT1, hOCT2, and hOCT3 were verified in functional assays and Western blots. For the uptake assay, 5 x 105 cells were seeded on 12-well plastic cell culture clusters. The uptake assay was performed after monolayer formation.
Plasmids. The plasmids pEXO-hOCT1 and pEXO-hOCT2, which contain the hOCT1 and hOCT2 cDNAs, respectively, were kindly provided by Dr. Kathleen M. Giacomini (University of California, San Francisco, CA). The hOCT1- and hOCT2-coding regions were amplified from these plasmids using polymerase chain reaction and Pfu DNA polymerase. The following specific primers were designed based on the nucleotide sequences for human OCT1 and OCT2 (GenBank accession nos. NM_003057 [GenBank] and NM_003058 [GenBank] ): hOCT1 forward, 5'-GCCGGTACCATGCCCACCGTGGATGACATTC-3' and reverse, 5'-GCCGGTACCTCAGGTGCCCGAGGGTTCTG-3'; and hOCT2 forward, 5'-GCCGGTACCATGCCCACCACCGTGGAC-3' and reverse, 5'-GCCGGTACCTTAGTTCAATGGAATGTCTAGTTTCTG-3' (KpnI restriction sites underlined). The isolated cDNAs were subcloned into the KpnI site of pcDNA3.1(–) (Invitrogen, San Diego, CA). The plasmid pcDNA 3.1(+)-hOCT3 (NM_021977 [GenBank] ) was kindly provided by Dr. Hitoshi Endou (Fuji Biomedix Co., Ltd., Tokyo, Japan).
Cellular Accumulation Assay. Before the cellular accumulation assay, the growth medium was removed from the cell cultures, and the attached cells were washed with Eagle's medium and preincubated in serum-free DMEM at 37°C for 1 h. The uptake study was initiated by the addition of 1 ml of medium that contained 200 nM [3H]MPP+ or [14C]TEA and 10 µM HPP+ or RHPP+ in the absence or presence of known hOCT inhibitors, i.e., 100 µM verapamil, 50 µM phenoxybenzamine, 50 µM cimetidine, or 50 µM corticosterone, at 37°C. The uptake study was stopped by three washes with 2 ml of ice-cold phosphate-buffered saline, and the cells were lysed with 100 µl of 1x cell culture lysis reagent (CCLR buffer; Promega, Madison, WI), which contained 100 mM potassium phosphate, pH 7.8, 1 mM EDTA, 7 mM 2-mercaptoethanol, 1% (v/v) Triton X-100, and 10% (v/v) glycerol. The levels of [3H]MPP+ and [14C]TEA radioactivity in the cell lysates were measured using the MicroBeta TriLux 96-well Scintillation/Luminescence Detector (Wallac Oy, Turku, Finland). For the measurement of HPP+ and RHPP+, the cells were lysed by the addition of 200 µl of acetonitrile/citrate buffer (90:10) (Zhang et al., 2002) and incubation at room temperature for 10 min, followed by shaking for 10 min. The cell lysates were transferred to microcentrifuge tubes on ice, and the supernatant was recovered after centrifugation at 13,000 rpm for 3 min. The concentrations of HPP+ and RHPP+ were determined by liquid chromatography-tandem mass spectrometry (LC/MS/MS).
Measurements of HPP+ and RHPP+ Using LC/MS/MS. The HPP+ and RHPP+ standards were dissolved in methanol at a concentration of 10 µM. These stock solutions were serially diluted with methanol to concentrations of 0.1, 0.5, 1, 5, 10, 20, 50, and 100 nM. The concentrations of HPP+ and RHPP+ in acetonitrile/citrate were quantified using liquid chromatography mass spectrometry with the PE SCIEX API 3000 LC/MS/MS system (Applied Biosystems, Foster City, CA), which was equipped with an electrospray ionization interface that was used to generate positive ions [M+H]+. The compounds were separated on a reversed-phase column (Luna C18, 5 mm x 50 mm internal diameter, 3-µm particle size; Phenomenex, Torrance, CA) with an isocratic mobile phase that consisted of acetonitrile and 5 mM ammonium acetate buffer (70:30). The mobile phase was eluted at 0.2 ml/min using an HP 1100 series pump (Agilent, Wilmington, DE). The turboion spray interface was operated in the positive ion mode at 5500 V and 350°C. The operating conditions, which were optimized by the flow injection of a mixture of all the analytes, were as follows: nebulizing gas flow, 1.04 l/min; auxiliary gas flow, 4.0 l/min; curtain gas flow, 1.44 l/min; orifice voltage, 80 V; ring voltage, 400 V; and collision gas (nitrogen) pressure, 3.58 x 10–5 Torr. The quantitation was performed by multiple reaction monitoring of the protonated precursor ion and the related product ion for HPP+ and RHPP+, using the internal standard method with peak area ratios and a weighting factor of 1/x. The mass transitions used for HPP+ and RHPP+ were m/z 354
165 and 356149, respectively (collision energy, 40 eV; dwell time, 200 ms). Quadrupoles Q1 and Q3 were set on unit resolution. The analytical data were processed using the Analyst software version 1.2 (Applied Biosystems). Most of the cell lysates were analyzed directly without dilution. One microliter of each lysate was injected into the column. For the 1st min of each run, the elution was diverted to waste. The HPP+ and RHPP+ concentrations in the lysates were within the standard curve range.
Western Blotting. Because the uptake of HPP+ and RHPP+ is influenced by the expression levels of hOCTs in the membrane, the intracellular concentrations of both metabolites were adjusted according to the hOCT expression levels. The levels of expressed hOCTs were determined by Western blotting. For the total protein extracts, transiently transfected or stably expressed cells of MDCK-hOCT1, MDCK-hOCT2, and MDCK-hOCT3 were lysed with one volume of radioimmunoprecipitation assay cell lysis buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris-Cl, pH 7.5) for 10 min. The samples were separated by SDS-polyacrylamide 4 to 12% gradient gel electrophoresis, and then electroblotted onto nitrocellulose membranes (Bio-Rad, Hercules, CA). The nitrocellulose membranes were incubated with 5% nonfat dry milk in Tris-buffered saline/Tween 20 for 2 h. The membranes were then incubated with polyclonal antibodies against OCT1, OCT2, and OCT3 (Santa Cruz Biotechnology, Santa Cruz, CA) and an anti-actin antibody (Cell Signaling Technology, Beverly, MA) overnight at 4°C. After rinsing with TBST (1.37 M NaCl, 0.3% Tween 20, 200 mM Tris Cl, pH 7.6), the membranes were incubated with horseradish peroxidase-labeled anti-goat IgG antibody or anti-rabbit IgG antibody (Santa Cruz Biotechnology) and visualized using the Enhanced ChemiLuminescence (ECL) System (Santa Cruz Biotechnology).
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Results |
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), hOCT-mediated cellular uptake of [3H]MPP+, which is a well known hOCT substrate, was determined in Caco-2 cells, as a positive control. Caco-2 cells were incubated with 200 nM [3H]MPP+ for various periods of time in the absence or presence of 100 µM verapamil, a known hOCT inhibitor. The cellular uptake of [3H]MPP+ increased nonlinearly for up to 90 min (Fig. 2A). In the presence of verapamil, the intracellular concentration of [3H]MPP+ in Caco-2 cells was significantly reduced by 2-fold within 90 min (Fig. 2A). When the haloperidol metabolites 10 µM HPP+ and RHPP+ were incubated under the same conditions as for [3H]MPP+, the intracellular concentrations of HPP+ and RHPP+ showed no proportional increases, although saturation was not complete until the cells were incubated for 90 min (Fig. 2, B and C). However, the intracellular accumulations of HPP+ and RHPP+ in Caco-2 cells were significantly reduced by approximately 2-fold in the presence of verapamil for 90 min (Fig. 2, B and C). The intracellular concentrations of HPP+ and RHPP+ were also significantly reduced by the addition of other inhibitors of OCT1, OCT2, and OCT3, i.e., phenoxybenzamine, cimetidine, and corticosterone (Fig. 3). Intracellular Accumulations of HPP+ and RHPP+ in hOCT1-, hOCT2-, and hOCT3-Expressing MDCK Cells. To further characterize hOCT-mediated uptake of HPP+ and RHPP+, the intracellular concentrations of these metabolites were evaluated in MDCK cells that transiently overexpressed hOCT1, hOCT2, and hOCT3. Twenty-four hours after transfection of the MDCK cells with each of the hOCTs, 200 nM [3H]MPP+, 10 µM HPP+, or 10 µM RHPP+ were added for 30 min at 37°C in the absence or presence of 50 µM phenoxybenzamine (Fig. 4). The activities of the overexpressed hOCTs were confirmed by the 2- to 3-fold higher [3H]MPP+ uptake into the hOCT1-, hOCT2-, and hOCT3-overexpressing cells compared with vector-transfected, control, mock-treated cells (Fig. 4A). The level of [3H]MPP+ uptake was significantly reduced in the presence of phenoxybenzamine in the hOCT-transfected MDCK cells, as well as in the mock-treated cells. The uptake of HPP+ was also increased by 2- to 3-fold in MDCK cells that overexpressed hOCT1, hOCT2, and hOCT3 (Fig. 4B), whereas RHPP+ uptake was not increased in MDCK cells that overexpressed hOCT2 (Fig. 4C). Furthermore, overexpression of the hOCT1, hOCT2, and hOCT3 proteins was verified by Western blotting (Fig. 4D). For the kinetic analysis of HPP+ and RHPP+ uptake, we established MDCK cells that stably expressed hOCT1, hOCT2, and hOCT3 and measured the uptake of HPP+ and RHPP+ by these cells. The functional expression of hOCT1, hOCT2, and hOCT3 was verified by [3H]MPP+ and [14C]TEA uptake assays (Fig. 5, A and B) and by Western blotting (Fig. 5C). The kinetics of HPP+ and RHPP+ uptake were examined by incubating the MDCK cells that stably expressed hOCTs with either 10 µM HPP+ or 10 µM RHPP+ at 37°C for various periods of time (1, 5, 10, 15, 30, 60, or 90 min) (Fig. 6, A and B). The rates of uptake of HPP+ and RHPP+ were nonlinear for up to 90 min in the MDCK cells that overexpressed hOCT1, hOCT2, and hOCT3. However, RHPP+ was not transported by hOCT2-overexpressing MDCK cells.
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The concentration dependencies of the uptake rates were studied by incubating hOCT1-, hOCT2-, and hOCT3-expressing MDCK cells with increasing concentrations (0, 0.1, 0.5, 1, 2, 3, 5, 10, and 15 µM) of HPP+ or RHPP+ for 15 min at 37°C (Fig. 7). From the uptake study, HPP+ accumulated in hOCT-overexpressing MDCK cells in a concentration-dependent manner, with estimated Km values of 0.99, 2.79, and 2.23 µM and Vmax values of 282.1, 256.1, and 400.2 pmol/min/µg protein for hOCT1, hOCT2, and hOCT3, respectively (Fig. 7, A–C) (Table 1). RHPP+ accumulated in the hOCT1- and hOCT3-overexpressing MDCK cells, with estimated Km values of 5.15 and 8.21 µM and Vmax values of 1230.9 and 1348.6 pmol/min/µg protein for hOCT1 and hOCT3, respectively (Fig. 7, D and E) (Table 1). These results suggest that both HPP+ and RHPP+ are substrates for hOCTs, with the exception of RHPP+ for hOCT2.
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Discussion |
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In the present study, we identified the transporters for HPP+ and RHPP+ as hOCTs. Haloperidol metabolites are structurally similar to the neurotoxin MPP+ (Fig. 1), which is an agent of neurodegeneration. Structurally, these compounds share a highly cationic pyridinium group. Functionally, they exhibit similar brain toxicities in terms of neuronal cell death, attributable to the inhibition of mitochondrial function. Initially, we investigated the transport of haloperidol metabolites into Caco-2 cells, which express various kinds of transporters and are able to transport organic cations across membranes via two distinct Na+-independent transporters, hOCT1 and hOCT3 (Martel et al., 2001). Caco-2 cells represent an appropriate model system for intestinal epithelial permeability studies, and the Caco-2 cell system offers considerable advantages for the study of the functional properties of transport processes compared with the use of intestinal tissues in vivo or in vitro (Martel et al., 2000). MPP+, which is a cationic compound, has also been identified as a substrate for hOCTs. Considering the similarities between MPP+ and haloperidol metabolites, we hypothesized that hOCTs mediate the transport of haloperidol metabolites. Therefore, we investigated the levels of accumulation of HPP+ and RHPP+ in Caco-2 cells and inhibited these transport processes using hOCT inhibitors.
The intracellular uptake of HPP+ and RHPP+ in Caco-2 cells showed nonlinear saturation (Fig. 2, B and C). Pretreatment with hOCT inhibitors, such as verapamil (Martel et al., 2000), cimetidine (Zhang et al., 1997; Grundemann et al., 1998), phenoxybenzamine, and corticosterone (Hayer-Zillgen et al., 2002), significantly reduced haloperidol metabolite uptake (Figs. 2 and 3). These compounds markedly inhibited hOCT function, although their effects on other transporters have not been fully investigated. Furthermore, phenoxybenzamine is a very specific inhibitor of hOCTs and has no known inhibitory effects on other transporters. Therefore, the inhibition of haloperidol metabolite uptake by these hOCT inhibitors suggests that hOCTs mediate the transport of these drugs.
The role of hOCTs in haloperidol metabolite uptake was demonstrated in MDCK cells that overexpressed hOCT1, hOCT2, or hOCT3. Cells that overexpressed hOCT1 and hOCT3 accumulated HPP+ and RHPP+, and the specificity of transport was verified by pretreatment with hOCT inhibitor (Fig. 4). HPP+ was accumulated to high levels in MDCK cells that stably expressed the hOCTs (Fig. 6). Interestingly, hOCT2-expressing cells accumulated HPP+ but not RHPP+. These results indicate that each hOCT has a different substrate spectrum.
Human organic cation transporter systems have been studied primarily in the kidney and liver because these organs play major roles in the elimination of xenobiotics (Pritchard and Miller, 1993; Ullrich, 1994). The hOCT1 and hOCT2 isoforms are primarily expressed in the liver and kidney, respectively, and to a lesser degree in the small intestine (Gorboulev et al., 1997; Busch et al., 1998; Zhang et al., 1998). These two hOCTs exhibit considerable overlap in terms of substrate specificity, as revealed by MPP+ and HPP+. The neuronal toxicity of haloperidol results from the toxic products of its metabolism, which is mediated mainly by CYP3A4 and partially by another P450 isoform, CYP2D6. CYP3A4 is highly expressed in the liver and intestine. Thus, haloperidol can be metabolized by CYP3A4 in hepatocytes and intestinal epithelia and then transported into the bile duct and bloodstream through the actions of hOCT1 and hOCT2. The metabolites can be eliminated in the urine through hOCTs, which are highly expressed in the kidney. The contributions of hOCT1 and hOCT2 to the hepatic, intestinal, and renal transport of haloperidol metabolites need to be studied further in vivo to develop a better understanding of haloperidol disposition.
Several cationic neurotoxins and neurotransmitters are good substrates for hOCT1 and hOCT2 (Pritchard and Miller, 1993; Ullrich, 1994) and, given the potential roles of these transporters in the handling of neurotoxins and neurotransmitters, attempts have been made to detect the expression of hOCT1 and hOCT2 in the brain (Grundemann et al., 1994; Gorboulev et al., 1997; Zhang et al., 1998). Although hOCT1 and hOCT2 mRNAs were not detected in the brain by Northern blot analysis (Gorboulev et al., 1997; Zhang et al., 1997), the results of studies using more sensitive reverse transcription-polymerase chain reaction methods have indicated that hOCT2-specific transcripts are present in the brain (Gorboulev et al., 1997; Grundemann et al., 1997). The neuronal expression of hOCT2 has been suggested by in situ hybridization and immunocytochemistry results (Busch et al., 1998). Assuming that hOCT2 is expressed in the blood-brain barrier, even at a low level, it may contribute to the distribution of haloperidol into the brain. Given that hOCT2 has been shown to transport HPP+ in an effective manner in the present study, the HPP+ formed in peripheral tissues may be taken up into the brain by the hOCT2 transporter.
Another hOCT isoform, hOCT3, was first identified as the corticosterone-sensitive extraneuronal catecholamine transporter and has been detected in the brain cortex as well as in liver and heart tissues (Grundemann et al., 1998). The ability of hOCT3 to interact with cationic neurotoxins and neurotransmitters and the expression of hOCT3 in the brain suggest that hOCT3 plays a role in the distribution of haloperidol metabolites into the brain. Considering that hOCT3 is an effective transporter of HPP+ and RHPP+, it is possible that hOCT3 contributes to the transport of these neurotoxic metabolites. Further in vivo studies are required to investigate whether hOCT3 mediates the distribution of haloperidol pyridinium metabolites into the brain.
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Footnotes |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: HPP+, 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxybutyl]pyridinium ion; RHPP+, 4-(4-(chlorophenyl)-1–4-(fluorophenyl)-4-hydroxybutyl-pyridinium; MPP+, 1-methyl-4-phenylpyridinium (N-[methyl-3H]-methyl-4-phenylpyridinium acetate); hOCT, human organic cation transporter; MDCK, Madin Darby canine kidney; TEA, tetraethylammonium; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; LC/MS/MS, liquid chromatography-tandem mass spectrometry; OCT, organic cation transporter.
Address correspondence to: Dr. Jae-Gook Shin, Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Department of Clinical Pharmacology, Busan Paik Hospital, 633-165 Gaegum-Dong, Jin-Gu, Busan 614-735, Korea. E-mail: phshinjg{at}inge.ac.kr
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75-kDa range.
