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Address correspondence and reprint requests to Hiroshi Takeda, Ph.D., Department of Pharrmacology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160–8402, Japan. E-mail: firstname.lastname@example.org
In this study, we examined the molecular and functional characterization of choline uptake into cultured rat cortical astrocytes. Choline uptake into astrocytes showed little dependence on extracellular Na+. Na+-independent choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis–Menten constant (Km) of 35.7 ± 4.1 µm and a maximal velocity (Vmax) of 49.1 ± 2.0 pmol/mg protein/min. Choline uptake was significantly decreased by acidification of the extracellular medium and by membrane depolarization. Na+-independent choline uptake was inhibited by unlabeled choline, acetylcholine and the choline analogue hemicholinium-3. The prototypical organic cation tetrahexylammonium (TEA), and other n-tetraalkylammonium compounds such as tetrabutylammonium (TBA) and tetrahexylammonium (THA), inhibited Na+-independent choline uptake, and their inhibitory potencies were in the order THA > TBA > TEA. Various organic cations, such as 1-methyl-4-tetrahydropyridinium (MPP+), clonidine, quinine, quinidine, guanidine, N-methylnicotinamide, cimetidine, desipramine, diphenhydramine and verapamil, also interacted with the Na+-independent choline transport system. Corticosterone and 17β-estradiol, known inhibitors of organic cation transporter 3 (OCT3), did not cause any significant inhibition. However, decynium22, which inhibits OCTs, markedly inhibited Na+-independent choline uptake. RT-PCR demonstrated that astrocytes expressed low levels of OCT1, OCT2 and OCT3 mRNA, but the functional characteristics of choline uptake are very different from the known properties of these OCTs. The high-affinity Na+-dependent choline transporter, CHT1, is not expressed in astrocytes as evidenced by RT-PCR. Furthermore, mRNA for choline transporter-like protein 1 (CTL1), and its splice variants CTL1a and CTL1b, was expressed in rat astrocytes, and the inhibition of CTL1 expression by RNA interference completely inhibited Na+-independent choline uptake. We conclude that rat astrocytes express an intermediate-affinity Na+-independent choline transport system. This system seems to occur through a CTL1 and is responsible for the uptake of choline and organic cations in these cells.
Choline is an organic cation that plays a critical role in the structure and function of biological membranes in all cells as an essential component of membrane phospholipids, phosphatidylcholine and sphingomyelin. Large degrees of choline uptake and phosphatidylcholine biosynthesis are necessary for new membrane synthesis. In the brain, choline plays an additional role as a precursor for the synthesis of the neurotransmitter, acetylcholine (Klein et al. 1993). Since the brain has only a limited capacity to synthesize choline de novo, most central nervous system choline is derived from the systemic circulation or from recycling from cerebral lipids (Wurtman 1992). Choline uptake is an important regulatory process in the brain, and high-affinity choline uptake has been previously shown to be the rate-limiting step in acetylcholine synthesis (Yamamura and Snyder 1972, 1973). Choline transport is demonstrated classically in two major systems that are classified by the degree of affinity for choline and Na+-dependency (Lockman and Allen 2002). The high-affinity and Na+-dependent system is localized in pre-synaptic cholinergic nerve terminals and is possibly coupled with acetylcholine synthesis. A low-affinity and Na+-independent system is found throughout various tissues, and this system primarily supplies choline for the synthesis of phosphatidylcholine and other phospholipids in the cellular membrane (Lockman and Allen 2002). The kinetics of choline transport in various cellular compartments of the brain are complex. For example, choline transport in the cholinergic synapse occurs via the high-affinity Na+-dependent choline transport system (Apparsundaram et al. 2000, 2001; Okuda et al. 2000), and choline transport in rat cortical synaptosomes occurs through high- and low-affinity Na+-independent transport mechanisms (Ferguson et al. 1991). Moreover, brain endothelial cells are found to express a high-affinity choline uptake system and are independent of extracellular Na+ (Sawada et al. 1999; Friedrich et al. 2001). Glial cells also contain choline transport systems that exhibit low-affinity uptake (Massarelli et al. 1974; Von Spreckelsen et al. 1988). However, the nature of the glial choline transport system is poorly understood.
Choline uptake is dependent upon carrier-mediated transport, since a charged cation under physiological pH does not cross cell membranes readily by passive diffusion. A high-affinity choline transporter, CHT1, has recently been cloned and characterized, and is thought to be unique to cholinergic neurons (Apparsundaram et al. 2000, 2001; Okuda et al. 2000). CHT1 is a Cl–- and Na+-dependent co-transporter that is sensitive to the choline analogue, hemicholinium-3, and is thought to be part of the rate-limiting step in acetylcholine synthesis. Indeed, the dynamic regulation of CHT1 in response to changes in cholinergic neuronal activity appears to match pre-synaptic acetylcholine synthesis to the rate of acetylcholine release. As an organic cation, choline is known to be a substrate for carriers of organic cation transporters (OCTs). To date, three different OCTs have been cloned and their function, which involves an Na+-independent uptake mechanism, has been characterized (Burckhardt and Wolff 2000; Koepsell et al. 2003). OCT1 and OCT2 are predominately expressed in the kidney, liver and, to a lesser extent, intestine (Gründemann et al. 1994, 1997; Okuda et al. 1996; Koepsell et al. 1999). The expression of OCT3 is widespread in rat tissues, including the brain (Wu et al. 1998; Takeda et al. 2002; Inazu et al. 2003a). These transporters recognize a multitude of endogenous and exogenous organic cations as substrates and exhibit considerable overlap in substrate specificity. Choline also interacts with these transporters with varying affinity. Another choline transporter, called choline transporter-like protein 1 (CTL1), has been cloned from Torpedo marmorata, and was first cloned as a suppressor for a yeast choline transport mutation from a Torpedo electric lobe yeast expression library by functional complementation (O'Regan et al. 2000; O'Regan and Meunier 2003). Rat CTL1 was also cloned as a homologous rat gene of the CTL protein family (O'Regan et al. 2000). CTL1 is expressed in several neuronal populations, including motor neurons, as well as in oligodendrocytes. Recently, two major rat splice variants of CTL1 (CTL1a and CTL1b) were identified; CTL1a and CTL1b are both expressed in oligodendrocytes, while CTL1a is also expressed alone in neuronal cell populations (Traiffort et al. 2005). However, little is known about the physiological function of CTL1 protein. The present study was undertaken to investigate the possible relationship between choline transport and the expression of the OCTs, CHT1 and CTL1 in rat cortical astrocytes.
Materials and methods
Sprague-Dawley rats from Charles River (Tokyo, Japan) were used for astrocyte cultures. Tissue culture media, kanamycin, media supplements and fetal calf serum were purchased from Gibco BRL (Gaithersburg, MD, USA). Cells were grown on plastic dishes, plates or flasks (Falcon, Becton Dickinson, NJ, USA). Dispase I (neutral protease; grade I) and DNase I were obtained from Roche (Mannheim, Germany). Choline, betaine, carnitine, hemicholinium-3 (HC-3), decynium22, tetraethylammonium chloride (TEA), tetrabutylammonium chloride (TBA), tetrahexylammonium chloride (THA), clonidine, quinine, quinidine, guanidine, N-methylnicotinamide (1-NMN), cimetidine, desipramine, diphenhydramine, corticosterone, 17β-estradiol, verapamil, 2-aminobicyclo-heptanecarboxylic acid (BCH), 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), alpha-(methylamino)isobutyric acid (MeAIB), vesamicol, 2-[N-morpholino]ethanesulfonic acid (MES), Triton X-100 and N-methyl-d-glucamine were obtained from Sigma Chemical Co. (St Louis, MO, USA). 1-Methyl-4-tetrahydropyridinium (MPP+) was purchased from Research Biochemicals International (Natick, MA, USA). Isogen was purchased from Nippon Gene (Tokyo, Japan). Omniscript Reverse Transcriptase and Qiagen Taq were purchased from Qiagen K.K. (Tokyo, Japan). RNase inhibitor and oligo(dT)16 primers were purchased from Perkin-Elmer Biosystems (Branchburg, NJ, USA). All of the siRNA duplexes (Stealth RNAi) were synthesized by Invitrogen Corporation (Carlsbad, CA, USA). Optifect transfection reagent and Opti-MEM I reduced serum medium were purchased from Invitrogen Corporation. Molecular ruler 100 bp DNA was purchased from Nippon Bio-Rad Laboratories (Tokyo, Japan). All other reagents were of analytical grade. [3H]Choline chloride (specific activity: 3182.0 GBq/mm) was obtained from Perkin Elmer Life Sciences, Inc. (Boston, MA, USA).
Astrocyte cultures were isolated from neonatal rat cerebral cortices as previously described (Inazu et al. 2001, 2003b). Cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37°C, and grown in Minimum Essential Medium supplemented with 10% (v/v) fetal calf serum and 20 mg/L kanamycin. The medium was changed every 2 days. After 10 days, rat astrocyte cultures were purified and re-plated into 24-well plates pre-coated with poly-d-lysine (Biocoat, Becton Dickinson, NJ, USA). Confluent 1- to 2-week-old cultures were used for further experiments. The purity of the astrocyte cultures was assessed by immunocytochemistry. In purified astrocyte cultures, > 98% of the cells were stained with antibodies (clone G-A-5) specific to glial fibrillary acid protein, an astrocyte marker. Neurons were not observed, as evidenced by a lack of staining for microtubule-associated protein-2 (MAP2) antibody.
[3H]Choline uptake into astrocytes
The growth medium was removed from the 24-well culture plates. The astrocyte cultures were washed twice with uptake buffer, consisting of 125 mm NaCl, 4.8 mm KCl, 1.2 mm CaCl2, 1.2 mm KH2PO4, 5.6 mm glucose, 1.2 mm MgSO4 and 25 mm HEPES adjusted to pH 7.4 with Tris, and 0.2 mL/well of uptake buffer was added. The astrocyte cultures were left to pre-incubate for 30 min at 37°C in humidified 5% CO2 and 95% air. The test compounds (0.025 mL) were added and incubated for 20 min at 37°C in humidified 5% CO2 and 95% air. This was followed by the addition of 0.025 mL [3H]choline. Uptake was terminated after 10 min by removal of the uptake buffer and three rapid washes with ice-cold uptake buffer. The cultures were dissolved in 0.8 mL 0.1 m NaOH and 0.1% Triton X-100, and aliquots were taken for liquid scintillation counting and protein assay. When Na+-free buffer was used, the Na+-free buffer was modified by replacing NaCl with an equimolar concentration of N-methyl-d-glucamine chloride. Uptake buffers of varying pH (pH 5.5, 6.5, 7.5 and 8.5) were prepared by mixing 25 mm MES/Tris (pH 5.5) and 25 mm Tris/HEPES (pH 8.5). Both buffers contained 125 mmN-methyl-d-glucamine chloride, 4.8 mm KCl, 1.2 mm CaCl2, 1.2 mm KH2PO4, 5.6 mm glucose and 1.2 mm MgSO4. Specific uptake of [3H]choline was calculated as the difference between total [3H]choline uptake in the presence and absence of 20 mm unlabeled choline. Protein concentrations were determined according to the method of Lowry et al. (1951) using a DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA).
RNA extraction and RT-PCR
Astrocyte cultures were washed with sterile Dulbecco's phosphate-buffered saline and RNA was extracted by the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi 1987). Total RNA (2 µg) was reverse-transcribed into cDNA using oligo(dT)16 primers and Omniscript Reverse Transcriptase. The reaction mixture was incubated at 37°C for 60 min and then at 93°C for 5 min, followed by rapid cooling at 4°C. The sequences of the specific primers for rat OCT1, OCT2, OCT3, CHT1, CTL1, CTL1a, CTL1b, β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as summarized in Table 1. The PCR reaction was performed using a TaKaRa PCR Thermal Cycler MP (Model TP3000 TaKaRa Biomedicals, Tokyo, Japan) as follows: 2 min at 94°C, followed by 35 cycles consisting of 0.5 min denaturation at 94°C, 0.5 min respective annealing temperatures and 1 min primer extension at 72°C. This was followed by extension at 72°C for 7 min. The PCR products were loaded on a 3% NuSieve agarose gel (Cambrex Bio Science Rockland, Inc., Rockland, ME, USA) pre-stained with 0.5 µg/mL ethidium bromide for electrophoresis in parallel with an appropriate DNA size marker.
Table 1. Primer sequences used for transporters detection
Both sense and antisense strands of the RT-PCR product were sequenced by primer walking. Sequencing by the dideoxynucleotide chain termination method was performed by Taq DyeDeoxy terminator cycle sequencing with an automated Perkin-Elmer Applied Biosystems 377 Prism DNA sequencer.
CTL1 small interfering RNA (siRNA) and siRNA transfections
The sequence (5′-GGGACTTCATGTTATTCCTAGGCAA-3′) was selected as the target region and corresponds to nucleotides 1609–1633 that are uniquely present in the rat CTL1 coding region (GenBank accession number AJ245619). To generate double-stranded CTL1 siRNA, the following oligonucleotides were synthesized by the Invitrogen Corporation: 5′-GGGACUUCAUGUUAUUCCUAGGCAA-3′ (sense); 5′-UUGCCUAGGAAUAACAUGAAGUCCC-3′ (antisense). The same nucleotides used to generate the siRNA probe were used in a scrambled sequence to generate a disordered siRNA (scr siRNA). This served as a control for non-specific effects due to the transfection of duplex RNA. Specifically, the sequences of the control oligonucleotides used were: 5′-GGGCUUGUAAUUCUUAUCGGACCAA-3′ (sense); and 5′-UUGGUCCGAUAAGAAUUACAAGCCC-3′ (antisense). Astrocytes were seeded onto 24-well tissue culture plates at 50–60% confluency. Culture medium was removed and the cells were washed once with Opti-MEM I reduced serum medium; they were then transfected with siRNA (50, 100 and 200 nm) using Optifect (3 µL/well) in Opti-MEM I reduced serum medium. After 24 h, an equal volume of fresh medium containing 20% serum was added. After an additional 48 h (i.e. 3 days after transfection), cells were used for analysis or experimentation.
The uptake experiments were routinely carried out in duplicate and each experiment was repeated three or four times. The results are expressed as means ± SE. The kinetic parameters and IC50 values were calculated by non-linear regression methods using the commercially available software Prism4 version 4.0b (GraphPad Software, Inc., San Diego, CA, USA). Ki values were derived from IC50 values as described by Cheng and Prusoff (1973). pKi values were calculated as the negative log of the corresponding Ki values expressed in molar concentrations. PCR products were visualized by UV illumination using Chemi Doc (Bio-Rad Laboratories). Statistical analysis was performed using the Mann–Whitney test for data from two groups and an analysis of variance (anova) followed by Dunnett's multiple comparison test for data from multiple groups. The results were considered statistically significant when p-values were less than 0.05 (two-tailed).
Time-course of [3H]choline uptake
We first examined the time course of [3H]choline uptake at a concentration of 10 nm in the presence and absence of Na+ in cultured rat astrocytes for 60 min (Fig. 1). [3H]Choline uptake in cultured rat astrocytes increased in a time-dependent manner; it was linear with time at least up to 10 min, and almost reached a plateau level at 45 min. The uptake of [3H]choline was mostly independent of extracellular Na+. Based on these findings, subsequent experiments were performed using an uptake period of 10 min in the absence of Na+.
Kinetics of Na+-independent [3H]choline uptake
The kinetic characteristics of Na+-independent [3H]choline uptake into cultured rat astrocytes were determined (Fig. 2). Astrocyte cultures were incubated for 10 min with [3H]choline at a concentration from 1.56 to 200 µm. Non-specific [3H]choline uptake was only 10% of the comparable value for total [3H]choline uptake. Kinetic analysis of Na+-independent [3H]choline uptake data (specific uptake), as computed by non-linear regression analysis, yielded a Michaelis–Menten constant (Km) of 35.7 ± 4.1 µm and a maximal velocity (Vmax) of 49.1 ± 2.0 pmol/mg protein/min. The Eadie–Hofstee plot (Fig. 2, inset) shows a single straight line (r2 = 0.8166, p = 0.0021), suggesting that Na+-independent [3H]choline uptake into cultured rat astrocytes is mediated by a single transport system.
Effects of membrane depolarization and extracellular pH on Na+-independent [3H]choline uptake
We investigated the electrogenicity of choline transport by examining the influence of membrane potential on Na+-independent [3H]choline uptake in rat astrocytes (Fig. 3a). The astrocyte membrane potential was depolarized either by increasing the concentration of K+ (108 mm) in the uptake buffer or by adding Ba2+ (10 mm) to the uptake buffer. Na+-independent [3H]choline uptake was significantly reduced when the astrocyte membrane potential was depolarized by either approach. We further investigated the influence of extracellular pH on Na+-independent [3H]choline uptake in rat astrocytes (Fig. 3b). Na+-independent [3H]choline uptake decreased dramatically when the extracellular pH was changed from 7.5 to 6.5 or 5.5. The uptake was enhanced approximately threefold when the pH of the extracellular medium was changed from 5.5 to 7.5. Na+-independent [3H]choline uptake could not be further stimulated by increasing the extracellular pH from 7.5 to 8.5.
Inhibitory effects of various organic cations on Na+-independent [3H]choline uptake
We investigated the inhibitory effects of various organic cations on the Na+-independent uptake of [3H]choline into rat astrocytes (Fig. 4a–d). The pKi values for the inhibition of Na+-independent [3H]choline uptake were calculated from the corresponding inhibition curves and are given in Table 2. The Na+-independent [3H]choline uptake into rat astrocytes was inhibited by unlabeled choline, HC-3 and acetylcholine. The prototypical organic cation, TEA, and other n-tetraalkylammonium compounds such as TBA and THA, inhibited Na+-independent [3H]choline uptake, and their inhibitory potencies were in the order THA > TBA > TEA. Various organic cations, such as MPP+, clonidine, quinine, quinidine, guanidine, 1-NMN, cimetidine, desipramine, diphenhydramine and verapamil, also interacted with the choline transport system in astrocytes. The correlations between the Ki values of some organic cations for Na+-independent [3H]choline uptake in astrocytes and those in the literature for rOCT1 and rOCT2 were not significant (vs. rOCT1: r = 0.4838, p < 0.8877; vs. rOCT2: r = 0.02867, p < 0.9333) (Table 2). Corticosterone and 17β-estradiol, known inhibitors of OCT3 (Hayer-Zillgen et al. 2002), and endogenous substances such as carnitine and betaine, did not cause any significant inhibition. However, the cyanine derivative, decynium22, a known inhibitor of OCTs (Hayer-Zillgen et al. 2002), markedly inhibited Na+-independent [3H]choline uptake. The selective inhibitors of the L- and A-type amino acid transporters BHC (1 mm) and MeAIB (1 mm), the selective organic anion transport inhibitor DIDS (1 mm) and the vesicular monoamine transporter inhibitor vesamicol (10 µm) did not affect Na+-independent [3H]choline uptake in rat astrocytes (data not shown).
Table 2. pKi and Ki values of various organic cations on Na+-independent [3H]choline uptake in rat astrocytes
Astrocyte pKi value ± S.E.
Astrocyte Ki value (μM)
rOCTl Ki value (μM)a
rOCT2 Ki value (μM)b
pKi values were calculated as the negative log of the coresponding Ki values expressed in molar concentrations. pKi values for the inhibition of Na+-independent [3H]choline uptake in astrocytes were calculated from the corresponding inhibition curves (Fig. 4). Each value represents the mean ± S.E. of four experiments. The Ki values for rOCTla and rOCT2b were taken from Koepsell et al. (1999), and Burckhardt and Wolff (2000). The correlations between the Ki values of various organic cations for the Na+-independent choline uptake in astrocytes and those in the litrature for rOCT 1 and OCT2 were not significant (vs. rOCTl: r=0.04838, p < 0.8877; vs. rOCT2: r=0.02867, p < 0.9333).
5.810 ± 0.015
4.752 ± 0.086
4.648 ± 0.027
4.573 ± 0.084
4.485 ± 0.079
4.080 ± 0.072
4.028 ± 0.023
3.991 ± 0.046
3.960 ± 0.042
3.881 ± 0.096
3.781 ± 0.421
3.765 ± 0.088
3.481 ± 0.149
2.998 ± 0.038
2.753 ± 0.055
2.585 ± 0.138
2.557 ± 0.043
RT-PCR demonstrates the expression of the mRNA for OCTs, CHT1, CTL1 and it splice variants, CTL1a and CTL1b
The expression of mRNA for OCT1, OCT2 and OCT3 was investigated by RT-PCR in RNA samples extracted from rat kidney and cortical astrocytes (Fig. 5a). The experimental conditions used for RT-PCR were validated by demonstrating RT-PCR products of the expected sizes for OCT1 (722 bp), OCT2 (960 bp) and OCT3 (851 bp) with rat kidney mRNA. As expected, mRNA for OCT1 and OCT2 was expressed abundantly in rat kidney. The expression of OCT3 mRNA in rat kidney was lower than that of mRNA for OCT1 and OCT2. Under similar conditions, mRNAs for all three OCTs were expressed at very low levels, but were still detectable, in rat astrocytes. The expression of OCT2 mRNA in rat astrocytes was lower than that of OCT1 and OCT3 mRNA. The RT-PCR data also show that rat astrocytes do not express the Na+-dependent high-affinity choline transporter CHT1 (Fig. 5b). RNA samples extracted from rat cortex, used as a positive control for CHT1 mRNA expression, yielded the expected RT-PCR product for CHT1 (445 bp). However, under similar conditions, the expression of CHT1 mRNA was not detectable in rat astrocytes.
In addition, we investigated the expression of CTL1 and its splice variants, CTL1a and CTL1b, in rat astrocytes. Expression of CTL1, CTL1a and CTL1b mRNA was investigated by RT-PCR in RNA samples extracted from rat cortical astrocytes and the rat frontal cortex (Fig. 5c). In rat astrocytes, CTL1 and CTL1a mRNAs were expressed abundantly, and while CTL1b mRNA was expressed at low levels, it was still detectable. Under similar conditions, mRNA for CTL1 and its splice variants, CTL1a and CTL1b, was expressed in rat frontal cortex. Sequence analysis demonstrated that the sequences of all RT-PCR products were identical to that of the respective gene.
Effects of CTL1 siRNA on CTL1 mRNA expression and Na+-independent [3H]choline uptake
We determined whether CTL1 knockdown induces any changes in the Na+-independent [3H]choline uptake of astrocytes (Fig. 6). CTL1 siRNA was used to decrease the expression of CTL1 mRNA, and expression levels were determined 3 days after siRNA transfection in rat astrocytes. RT-PCR analysis demonstrated that CTL1 siRNA significantly decreased the expression level of CTL1 mRNA, compared with that of scr siRNA, in a concentration-dependent manner (Figs 6a and b). Under similar conditions, the inhibition of CTL1 expression by CTL1 siRNA significantly inhibited Na+-independent [3H]choline uptake into astrocytes in a concentration-dependent manner (Fig. 6c). Na+-independent [3H]choline uptake was not affected when astrocytes were transfected with scr siRNA.
Choline is well known to be an acetylcholine precursor in the brain. After acetylcholine is released into the synaptic cleft, it is then hydrolyzed to choline and acetic acid by acetylcholinesterase and butylylcholinesterase, which are expressed in astrocytes. Choline is then free to be taken up into pre-synaptic nerve terminals for recycling. Thus, the supply of choline is essential for normal cholinergic nerve function. Since choline is a charged hydrophilic cation under physiological conditions, it cannot appreciably diffuse across cellular membranes in required quantities. As a result, choline transport is an important part of cellular membrane construction and is the rate-limiting step for physiological cholinergic neurotransmission. Two choline uptake systems, an Na+-dependent high-affinity choline uptake system responsible for the synthesis of acetylcholine and an Na+-independent low-affinity choline uptake system responsible for the synthesis of phospholipids, are known to exist in the brain (Haga and Noda 1973; Yamamura and Snyder 1973; Xin and Wightman 1997). The Na+-dependent high-affinity choline transporter, designated CHT1, has recently been cloned (Okuda et al. 2000). CHT1 is a known choline transporter that has been described at cholinergic neuronal membranes. However, the molecular identity of the Na+-independent low-affinity choline transporter has not yet been identified.
In this study, we found that astrocytes take up [3H]choline by an active saturable process that involves Na+-independent uptake mechanisms. The Km of Na+-independent [3H]choline uptake in astrocytes was 35.7 µm, suggesting the existence of an intermediate-affinity choline uptake system in astrocytes. The kinetic characteristics and Na+ dependency of the choline transport system in astrocytes are very similar to those previously reported for the mouse brain capillary endothelial cell line, MBEC4 (Kt = 20 µm reported by Sawada et al. 1999), for the immortalized rat brain microvessel endothelial cell line, RBE4 (Kt = 23 µm reported by Friedrich et al. 2001), and for the immortalized human keratinocyte cell line, HaCaT (Kt = 14.8 µm reported by Hoffmann et al. 2002). These reports have shown conclusively that choline is taken up into these cells by a specific transporter that is not OCT1, OCT2, OCT3 or CHT1. The expression of high-affinity (Km < 10 µm) Na+-dependent choline transporter CHT1 is not found in these cell lines or rat astrocytes. Interestingly, a very similar pH profile (pH-dependent uptake) was observed for choline transport in RBE4 and HaCaT cells. [3H]Choline uptake in astrocytes was significantly decreased when the pH of the uptake medium was changed from 7.5 to 5.5, indicating that the transport of choline is proton-dependent. These data suggest that [3H]choline may be transported by an organic cation/H+ exchanger. Moreover, Na+-independent [3H]choline uptake in astrocytes was markedly reduced when the astrocyte membrane potential was depolarized by increasing the concentration of K+ in the uptake buffer or by adding 10 mm Ba2+ to the uptake buffer. These findings support the notion of electrogenically facilitated transport of choline in astrocytes. Due to its positive charge at physiological pH, the rate of choline movement across cell membranes would be expected to be affected by changes in the membrane potential. These data show that the astroglial choline transporter resembles the OCT family with regard to its functional characteristics, as well as its dependence on membrane potential and pH, and its independence of extracellular Na+.
To date, three different potential-sensitive organic cation transporters have been cloned and characterized: OCT1, OCT2 and OCT3 (Gründemann et al. 1994, 1997, 1998; Okuda et al. 1996; Kekuda et al. 1998; Wu et al. 2000; Koepsell et al. 2003). All belong to the solute carrier family 22 (SLC22) of transporters, and most have been cloned from kidney and liver tissues (Koepsell 1998; Koepsell and Endou 2004). OCT1 and OCT2 accept choline as a substrate with comparatively low affinity (Gorboulev et al. 1997). The transport process is Na+-independent and has a Kt value of 620 µm in the case of OCT1 and 210 µm in the case of OCT2. However, OCT3 does not recognize choline as a substrate (Gründemann et al. 1998, 1999; Kekuda et al. 1998; Wu et al. 2000). Thus, we examined pharmacologically whether Na+-independent [3H]choline uptake into astrocytes is mediated by OCT1, OCT2 or OCT3. Decynium22, a potent cyanine-related inhibitor of OCTs (Rüss et al. 1993; Hayer-Zillgen et al. 2002), was effective in inhibiting Na+-independent [3H]choline uptake, with a Ki value of 1.55 µm. Decynium22 was first described as a potent inhibitor of OCT3 in the human cell line, Caki-1 (Ki value: 16 nm; Rüss et al. 1993), and was later shown to inhibit rat OCT1 (IC50 value: 0.36 µm; Gründemann et al. 1994) and rat OCT2 (IC50 value: 0.58 µm; Koepsell et al. 1999), albeit with about a 20- to 30-fold lower potency. The Ki value for the inhibition of Na+-independent [3H]choline uptake into astrocytes by decynium22 is much higher than the values in the literature. TEA, a prototypical organic cation that is very often used as a high-affinity reference compound for OCTs, has Ki values for OCT1 and OCT2 of 46.6 µm and 52.2 µm, respectively (Urakami et al. 1998). In this study, TEA interacted with the Na+-independent choline transport system with a very low affinity (Ki value: 2.77 mm). Interestingly, a previous report showed that n-tetraalkylammonium compounds with longer alkyl chain lengths (i.e. more hydrophobic and bulkier) were more potent inhibitors of human OCT1 (Zhang et al. 1999). We found that TEA, and other n-tetraalkylammonium compounds such as TBA and THA, inhibited Na+-independent [3H]choline uptake, and their inhibitory potencies were in the order THA (alkyl chain length: n = 6) > TBA (n =4) > TEA (n = 2). These results show that there is a good correlation between Ki values and the alkyl chain length of n-tetraalkylammonium compounds. These data indicate that the longer the alkyl chain length (i.e. more hydrophobic and bulkier), the higher the affinity of the n-tetraalkylammonium compounds for the transport of choline in astrocytes. To understand the effects of various organic cations on the uptake of [3H]choline by rat astrocytes further, an additional study was performed. In the present study, Na+-independent [3H]choline uptake was inhibited by various organic cations such as MPP+, clonidine, quinine, quinidine, guanidine, 1-NMN, cimetidine, desipramine, diphenhydramine and verapamil. There was no correlation between the Ki values for the inhibition of Na+-independent [3H]choline uptake in astrocytes by some organic cations and the literature values for OCT1 or OCT2. These data clearly show that the Na+-independent choline uptake in astrocytes does not occur via OCT1 and OCT2. OCT3 is known to be selective and potently inhibited by corticosterone and 17β-estradiol (Hayer-Zillgen et al. 2002; Inazu et al. 2003a). However, both agents failed to inhibit Na+-independent [3H]choline uptake in astrocytes, indicating that [3H]choline was also not transported by OCT3. It is well known that OCT3 does not recognize choline as a substrate. Furthermore, in this study, RT-PCR data show that mRNA for OCT1, OCT2 and OCT3 was expressed at very low levels in rat astrocytes. The present study has shown that Na+-independent [3H]choline uptake into rat astrocytes was not mediated by OCT1, OCT2 or OCT3. However, this system seems to exhibit OCT-like properties. Next, we examined the involvement of L- and A-type amino acid transporters, organic anion transporter and vesicular monoamine transporter on Na+-independent choline uptake in astrocytes. The results showed that these transporters also did not play a role.
Next, we searched for a new transporter as a candidate that would be an Na+-independent choline transport system. Recently, a novel family of transporters, called choline transporter-like (CTL) proteins, has been cloned from Torpedo marmorata (O'Regan et al. 2000; O'Regan and Meunier 2003). Rat CTL1 was also cloned as a homologous rat gene of the CTL protein family and exhibited saturable, HC-3-inhibitable (Ki value > 10 µm) and weak Na+-dependent uptake of choline (O'Regan et al. 2000; Traiffort et al. 2005). These uptake properties of CTL1 are very similar to those of the astroglial choline transporter, since the Ki value of HC-3 in the Na+-independent uptake of choline in rat astrocytes was 32.73 µm as observed in the present study and thus, these potencies are very similar. However, the pharmacological characteristics of CTL1 are not well known. In situ hybridization in rat tissues revealed the strong expression of rat CTL1 in motor neurons and oligodendrocytes, and, to a lesser extent, in various neuronal populations throughout the rat brain. Outside the central nervous system, high levels of rat CTL1 mRNA were detected in the mucosal cell layer of the colon. Recently, two major rat splice variants of CTL1 (CTL1a and CTL1b) were identified and CTL1a and CTL1b are both expressed in oligodendrocytes, while CTL1a is also expressed in neuronal cell populations (Traiffort et al. 2005). We confirmed the expression of mRNA for CTL1 and its splice variants in rat astrocytes. We found that the mRNA of both CTL1a and CTL1b was expressed in rat astrocytes, and that of CTL1a is abundantly expressed. The CTL1a- and CTL1b-induced choline transport displayed similar affinities for choline and HC-3 (Traiffort et al. 2005). The Km values for CTL1a and CTL1b were 34.7 and 33.4 µm, which were very similar to the Km values of Na+-independent [3H]choline uptake in astrocytes (35.7 µm). In the present study, we examined whether Na+-independent [3H]choline uptake in astrocytes is mediated by CTL1 using RNA interference. The inhibition of CTL1 mRNA expression by CTL1 siRNA completely inhibited Na+-independent [3H]choline uptake. These findings strongly suggest that CTL1 is functionally expressed in rat astrocytes and that it is responsible for Na+-independent choline uptake in these cells, since the strong expression of CTL1 in oligodendrocytes, the myelin-synthesizing cells in the brain, which require high levels of choline to synthesize phosphatidylcholine and sphingomyelin (O'Regan et al. 2000; Meunier and O'Regan 2002; Traiffort et al. 2005), suggests that CTL1 is involved in activated choline uptake for membrane synthesis in glial cells. Interestingly, CTL1 is expressed in cholinergic neurons, such as the motor neurons of the spinal cord and the facial nucleus (Traiffort et al. 2005), which is compatible with a role analogous to the plasma membrane high-affinity choline transporter CHT1. Brain astrocytes are located around synapses and at the periphery of capillaries, and their astroglial uptake systems are likely to play an important role in regulating the neuronal environment. The uptake of choline by astrocytes might serve as a local mechanism for regulation of the choline concentration in the extracellular space. However, the physiological role of CTL1 in the cholinergic nervous system is not well understood.
In conclusion, the present results suggest that rat astrocytes express an intermediate-affinity Na+-independent choline transport system. This system seems to occur through a CTL1 and may also play a key role in the disposition of choline and organic cations in these cells. Further studies are needed to elucidate the mechanisms that regulate glial CTL1 expression and translocation by astrocytes, and the interaction between glial CTL1 and neuronal CTL1 in the brain.
This work was supported by Grant-in-Aid no. 16047227 for Scientific Research on Priority Areas on the ‘Elucidation of glia-neuron network-mediated information processing systems’ from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and by ‘High-Tech Research Center’ Project for Private Universities: matching fund subsidy from the MEXT (2003–2007).