Functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, a human blood–brain barrier model
© Shimomura et al.; licensee BioMed Central Ltd. 2013
Received: 19 September 2012
Accepted: 22 January 2013
Published: 26 January 2013
Knowledge of the molecular basis and transport function of the human blood–brain barrier (BBB) is important for not only understanding human cerebral physiology, but also development of new central nervous system (CNS)-acting drugs. However, few studies have been done using human brain capillary endothelial cells, because human brain materials are difficult to obtain. The purpose of this study is to clarify the functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, which has been recently developed as an in vitro human BBB model.
Diphenhydramine, [3H]pyrilamine and oxycodone were used as cationic drugs that proved to be H+/OC antiporter substrates. The in vitro uptake experiments by hCMEC/D3 cells were carried out under several conditions.
Diphenhydramine and [3H]pyrilamine were both transported into hCMEC/D3 cells in a time- and concentration-dependent manner with Km values of 59 μM and 19 μM, respectively. Each inhibited uptake of the other in a competitive manner, suggesting that a common mechanism is involved in their transport. The diphenhydramine uptake was significantly inhibited by amantadine and quinidine, but not tetraethylammonium and 1-methyl-4-phenylpyridinium (substrates for well-known organic cation transporters). The uptake was inhibited by metabolic inhibitors, but was insensitive to extracellular sodium and membrane potential. Further, the uptake was increased by extracellular alkalization and intracellular acidification. These transport properties are completely consistent with those of previously characterized H+/OC antiporter in rat BBB.
The present results suggest that H+/OC antiporter is functionally expressed in hCMEC/D3 cells.
KeywordsHuman blood–brain barrier Human BBB model cell hCMEC/D3 cells Proton-coupled organic cation antiporter Organic cation transporter Transport function Diphenhydramine Pyrilamine Oxycodone Active transport Real-time PCR
The human brain is protected by the existence of the blood–brain barrier (BBB), which consists of brain capillary endothelial cells linked with tight junctions . It is well established that the polarized expression of numerous transporters and receptors at the brain capillary endothelial cells controls the blood–brain exchange of nutrients, waste products produced from neurotransmitter substances, and drugs . Therefore, knowledge of the molecular basis and transport function of the human BBB is important for not only understanding human cerebral physiology, but also for development of new central nervous system (CNS)-acting drugs. However, few studies have been done using human brain capillary endothelial cells, because human brain materials are difficult to obtain. In addition, isolation and primary culture of brain capillary endothelial cells are laborious and time-consuming procedures [3, 4]. Therefore, the development of simple in vitro BBB models is highly desirable.
Human immortalized brain capillary endothelial cells (hCMEC/D3) have recently been developed as an in vitro human BBB model . This cell line has been now extensively validated by numerous laboratories worldwide in pharmacological, toxicological, immunological and infection studies. These hCMEC/D3 cells retain many of the morphological and functional characteristics of the human BBB in terms of expression of multiple transporters, receptors, tight junction proteins and various ABC transporters, including ABCB1 (MDR1/P-gp), ABCC1 (MRP1), ABCC4 (MRP4), ABCC5 (MRP5), and ABCG2 (BCRP) [2, 6, 7]. Furthermore, several solute carrier (SLC) transporters responsible for the blood–brain exchange of mainly nutrients, including SLC2A1 (GLUT1), SLC16A1 (MCT1), SLC29A1 (ENT1) and so on, are highly expressed at the mRNA level in this cell line . On the other hand, little is known concerning the expression and function of influx transporters that may regulate the brain distribution of drugs, except for relatively abundant expression of SLCO2A1 (OATP2A1) at the mRNA level .
Recently, we have reported that a H+/OC antiporter is functionally expressed in the in vivo rat BBB and also in a conditionally immortalized rat BBB cell line (TR-BBB13 cells) [9, 10]. This H+/OC antiporter mediates blood–brain transport of CNS-acting cationic drugs such as pramipexole, oxycodone and diphenhydramine, in addition to pyrilamine, in rats. A brain microdialysis study revealed that this transporter actively transports oxycodone and diphenhydramine into the brain, and their unbound concentration in brain interstitial fluid (ISF) is 3- to 5-fold higher than that in blood [10, 11]. There is also evidence that clonidine  and methylenedioxymethamphetamine (MDMA)  are transported by H+/OC antiporter in the BBB and in peripheral cell lines, respectively. Although the molecular entity of this transporter remains unknown, the known substrates are secondary or tertiary amines with positive charge at physiological pH. This suggests that many CNS drugs used in the clinical setting may be efficiently taken up into the brain via the H+/OC antiporter at the BBB. In addition, this putative transporter is a potential target in the development of new CNS drugs.
The purpose of this study, therefore, is to clarify the functional expression of the H+/OC antiporter in hCMEC/D3 cells. We also discuss whether or not the results of in vitro uptake study using hCMEC/D3 cells can be extrapolated to the human BBB in vivo, as well as the relevance of our findings to cerebral physiology and to the development and proper use of CNS-acting cationic drugs.
Diphenhydramine hydrochloride was purchased from Wako Pure Chemical Industries (Osaka, Japan), [3H]Pyrilamine (23 – 30 Ci/mmol) and [14C]inulin (2.06 mCi/g) were purchased from Amersham Bioscience (Buckingshamshire, UK) and PerkinElmer Life and Analytical Sciences, Inc. (Walthan, Massachusetts, USA), respectively. Oxycodone was kindly provided by Takeda Pharmaceutical Co. Ltd. (Osaka, Japan). All other chemicals and reagents, including diphenhydramine, were commercial products of reagent grade.
The hCMEC/D3 cells had been immortalized by lentiviral transduction of the catalytic subunit of human telomerase and SV40-T antigen . The cells were cultivated at 37°C in EBM-2 medium (Takara Bio, Shiga, Japan) supplemented with 2.5% fetal bovine serum, 0.025% VEGF, 0.025% R3-IGF, 0.025% hEGF, 0.01% hydrocortisone, 5 μg/mL bFGF, 1% penicillin-streptomycin and 10 mM HEPES on rat collagen type I coated dishes in 95% air and 5% CO2.
Adult male Wistar rats weighing about 350 g were purchased from Japan SLC (Shizuoka, Japan); they were housed, three or four per cage, in a laboratory with free access to food and water and were maintained on a 12-hr dark/12-hr light cycle in a room with controlled temperature (24 ± 2°C) and humidity (55 ± 5%). This study was conducted according to guidelines approved by the Experimental Animal Ethical Committee of Teikyo University.
The hCMEC/D3 cells used for the experiments were between passage 25 and 35. The cells were seeded on rat collagen I-coated multi-well plates (Becton Dickinson) at a density of 0.2 × 105 cells/cm2. At 3 days after seeding the cells reached confluence, the cells were washed twice with 2 mL of phosphate-buffered saline (pH 7.4) and preincubated with the transport buffer (122 mM NaCl, 3 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 1.4 mM CaCl2, 10 mM D-glucose, 10 mM HEPES, pH 7.4) for 20 min at 37°C. After preincubation, 1 mL of the transport buffer containing diphenhydramine (30 μM) or [3H]pyrilamine (74 kBq/μL, 90 nM) was added to initiate uptake. The cells were incubated at 37°C for a designated time, and then washed three times with 2 mL of ice-cold incubation buffer to terminate the uptake. In the case of diphenhydramine, the cells were collected in 400 μL of 0.5% KH2PO4 solution, and stored at −20°C until HPLC determination as described below. For the determination of [3H]pyrilamine radioactivity, the cells were solubilized with 1 M NaOH for 60 min and the radioactivity was measured using a liquid scintillation counter after the addition of scintillation cocktail Hionic Fluor (PerkinElmer Life and Analytical Sciences). Cellular protein content was determined with a BCA protein assay kit (Pierce Chemical Co., Rochford, IL, USA).
where V is the initial uptake rate of substrate (nmol/min/mg protein), s is the substrate concentration in the medium (μM), Km is the Michaelis-Menten constant (μM) and Vmax is the maximum uptake rate (nmol/min/mg protein). Vmax/Km (pmol/min/mg protein/μM = μL/min/mg protein) values were calculated as the uptake clearance for the saturable transport component.
In order to examine the energy dependency of diphenhydramine uptake by hCMEC/D3 cells, the uptake was measured as described above after pretreatment with 25 μM rotenone (dissolved in the transport medium containing 0.25% ethanol) or 0.1% NaN3 for 20 min. In this experiment, 10 mM D-glucose in the transport medium was replaced with 10 mM 3-O-methylglucose to reduce metabolic energy. In order to examine the sodium requirement of diphenhydramine uptake by hCMEC/D3 cells, sodium ions were replaced with N-methylglucamine+. To examine the effects of reducing the membrane potential and proton gradient on diphenhydramine uptake by hCMEC/D3 cells, 10 mM valinomycin and 10 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, a protonophore) (dissolved in the transport medium containing 0.22% ethanol), respectively, were added to the transport medium. These studies were performed in parallel with controls in the presence of the corresponding ethanol concentration. Uptake was also measured at medium pH values of 6.0, 7.4 and 8.4. When the influence of intracellular pH (pHi) was examined, the uptake was measured in the presence of 30 mM NH4Cl to elevate pHi [9, 10]. To measure the uptake at acidic pHi, extracellular NH4Cl was removed after the preincubation with 30 mM NH4Cl, because intracellular NH3 rapidly diffuses out of the cells, resulting in the accumulation of protons released from NH4+ during NH3 generation in the cells. In the inhibition study, the uptake was measured after incubation with diphenhydramine (30 μM) for 15 sec in the presence of selected inhibitors (amantadine, quinidine, TEA, serotonin, MPP+ and choline) at the concentration of 1 mM. Diphenhydramine uptake (30 – 500 μM, 15 sec) was measured in the absence and presence of pyrilamine (150 μM) or oxycodone (500 μM). 3H]pyrilamine uptake (6 – 500 μM, 10 sec) was also measured in the absence and presence of diphenhydramine (50 μM).
In situ brain perfusion study
Brain perfusion was performed by the same method as reported previously [9, 14]. In brief, each rat was anesthetized and the right carotid artery was catheterized with polyethylene tubing (SP-10) filled with sodium heparin (100 IU/mL). The perfusate (Krebs-Henseleit buffer, 118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3, 1.2 mM KH2PO4, 2.5 mM CaCl2, 1.2 mM MgSO4, 10 mM D-glucose, pH 7.4) containing diphenhydramine (10 μM) or 3H]pyrilamine and 14C]inulin (0.9 μM), a brain intravascular marker, was passed through the catheter at the rate of 4.9 mL/min with an infusion pump (Harvard Apparatus, South Natick, MA, USA). After the infusion pump is started, 5.0 sec is required to fill the external carotid artery cannula . Therefore, 5.0 sec was routinely subtracted from the gross perfusion time in each experiment, to obtain the uptake time for which the perfusate was actually within the brain capillaries. At the end of uptake for 5 – 30 sec, rats were decapitated, and the right cerebral hemisphere was dissected from the perfused brain and weighed. The brain samples were stored at −20°C until determination of diphenhydramine. The brain uptake of 3H]pyrilamine (1.1 nM) was also determined as described above. The radioactivity was measured using a liquid scintillation counter as described above, after solubilization of the cerebral hemisphere in Soluene-350 (PerkinElmer Life and Analytical Sciences, Boston, MA, USA) at 55°C for 3 h and decolorization by addition of 0.3 mL of 30% H2O2. The value of the permeability-surface area product (PSBBB,inf), which represents in vivo BBB permeability, was calculated after correcting for remaining intravascular diphenhydramine or 3H]pyrilamine, estimated from the apparent brain uptake of 14C]inulin [9, 14].
Determination of diphenhydramine
The collected cells in 0.5% KH2PO4 solution were homogenized by sonication. For brain tissue, the cerebral hemisphere was homogenized in 5 volumes of 0.5% KH2PO4 solution. To the homogenate (300 μL) was added orphenadrine hydrochloride (50 μM, 6.3 μL) as an internal standard, as well as 60 μL of saturated K2CO3. After mixing, samples were extracted with 1.5 mL of hexane-isopropanol (98:2, v/v) by shaking on a horizontal shaker for 15 min and then centrifuged for 10 min at 800 g. The upper organic layer was transferred into a tube containing 300 μL of 0.5% KH2PO4. The tube was shaken for 15 min and centrifuged for 10 min at 800 g. The upper organic layer was aspirated off, then 60 μL of saturated K2CO3 was added to the lower aqueous phase and the extraction step and back-extraction step were repeated. The final K2CO3 solution phase was extracted again with 1.5 mL of hexane-isopropanol and the extract was dried under a nitrogen stream. The residue was reconstituted in 100 μL of mobile phase.
Diphenhydramine was determined by ultra-performance liquid chromatography (UPLC®) with a UV detector by modification of previously reported methods [16, 17]. A 7.5 μL aliquot was injected into the UPLC®. The UPLC® system (Waters ACQUITY, Milford, MA, USA) consisted of a binary solvent manager, sample manager and UV detector. The analytical column used was an ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm particle size, Waters). The UPLC separation was carried out at a flow rate of 0.15 mL/min with a mobile phase containing 25% acetonitrile and 0.22 M phosphate buffer. UV detection was performed at 205 nm. The retention times of diphenhydramine and orphenadrine were 6.4 and 9.1 min, respectively. The detection limit for quantification of diphenhydramine was 75 pmol.
Expression profiling of organic cation transporters by real-time PCR
Sense and antisense primers for quantitative PCR
Statistical analysis of the data was performed by employing Student’s t-test and by one-way analysis of variance followed by Dunnett’s test for single and multiple comparisons, respectively. Differences were considered statistically significant at P < 0.05.
Uptake kinetics of diphenhydramine and [3H]pyrilamine by hCMEC/D3 cells
Metabolic energy and ion dependence of the uptake of diphenhydramine by hCMEC/D3 cells
Effects of metabolic inhibitors (rotenone and sodium azide), protonophore (FCCP), sodium replacement and change in membrane potential by valinomycin on diphenhydramine uptake by hCMEC/D3 cells
Relative uptake (% of control)
Rotenone (25 μM) a),b)
29.6 ± 26.9 **
Sodium azide (0.1%) b)
11.8 ± 0.4 **
10 μM FCCP
30.6 ± 1.3 ***
95.3 ± 4.5
Valinomycin (10 μM) c)
84.1 ± 0.2
Inhibition of uptake of diphenhydramine and [3H]pyrilamine by hCMEC/D3 cells
Inhibitory effects of various compounds on diphenhydramine uptake by hCMEC/D3 cells
Relative uptake (% of control)
23.7 ± 3.8 ***
24.0 ± 6.2 ***
98.9 ± 12.3
84.7 ± 14.9
90.2 ± 11.4
74.1 ± 7.3
mRNA expression of organic cation transporters in hCMEC/D3 cells
mRNA expression levels of organic cation transporters in hCMEC/D3cells determined by quantitative RT-PCR analysis
(target mRNA x 105/GAPDH mRNA)
1.99 ± 0.21
8.59 ± 0.55
80.7 ± 4.6
248 ± 7.3
0.29 ± 0.02
1.15 ± 0.16
34.0 ± 2.4
In vivo blood-to-brain transport of diphenhydramine and [3H]pyrilamine in rats
The brain uptakes of diphenhydramine and 3H]pyrilamine were measured by the in situ brain perfusion technique. The brain/perfusate (B/P) ratios of diphenhydramine and 3H]pyrilamine linearly increased with increasing perfusion time up to 30 sec. The PSBBB,inf values for diphenhydramine and 3H]pyrilamine were 4.40 and 1.30 mL/min/g brain, respectively. The PSBBB,inf value for 3H]pyrilamine was similar to the value reported previously (1.6 mL/min/g brain) .
There is increasing evidence that the immortalized human brain capillary endothelial cell line hCMEC/D3 is a good model to predict BBB permeability in the human brain [5, 19]. In the present study, we further investigated the utility of this model by examining whether H+/OC antiporter is functionally expressed in the cells. Although this putative H+/OC antiporter still remains to be identified at the molecular level, it is known to transport several CNS-acting drugs with secondary or tertiary amine moieties, including diphenhydramine, oxycodone, pyrilamine and clonidine [9, 10, 12].
Diphenhydramine, pyrilamine and oxycodone each form a cation at physiological pH because they are weak bases having a tertiary amine moiety. We previously showed that diphenhydramine, pyrilamine and oxycodone are taken up via a pH-sensitive, energy-dependent, proton-coupled antiport system in TR-BBB13 cells, which are an in vitro rat BBB model [9, 10]. In addition, both diphenhydramine and oxycodone have been reported to be actively taken up by the brain across the BBB with a Kp,uu (unbound concentration ratio of brain interstitial fluid to plasma) value of more than 3 in rats [10, 11]. Consequently, we have suggested that H+/OC antiporter works in the rat BBB as an active influx transporter. In the present study, diphenhydramine and pyrilamine were used as substrates to investigate this activity.
The uptakes of diphenhydramine and 3H]pyrilamine were time- and concentration-dependent. Kinetic analyses revealed that the calculated uptake clearances (Vmax/Km) for diphenhydramine (220 μL/min/mg protein) and 3H]pyrilamine (184 μL/min/mg protein) were in good agreement with those of diphenhydramine (440 μL/min/mg protein) and 3H]pyrilamine (140 μL/min/mg protein) in conditionally immortalized rat brain capillary endothelial cells (TR-BBB13) [9, 10]. These results suggest that a transporter with similar transport activity and transport clearance to those observed in the case of TR-BBB13 cells is involved in uptake of diphenhydramine and 3H]pyrilamine by hCMEC/D3 cells. Furthermore, the transporter seems to show no marked species difference between TR-BBB13 cells and hCMEC/D3 cells.
The uptake of diphenhydramine was significantly inhibited by pretreatment with metabolic inhibitors, but was insensitive to extracellular sodium and membrane potential in hCMEC/D3 cells (Table 2), suggesting the involvement of a transporter having similar energy and membrane potential dependencies to those of TR-BBB13 cells. Furthermore, the diphenhydramine uptake by hCMEC/D3 cells showed pH-dependency characteristic of a proton-coupled antiporter. The uptake was increased at higher extracellular pH (pH 8.4), and decreased in the presence of FCCP. Intracellular acidification induced with NH4Cl, stimulated the uptake (Figure 3 and Table 2). As the pK a value of diphenhydramine is 8.98, the proportion of uncharged diphenhydramine can be estimated to be 20.8% at pH 8.4, 2.6% at pH 7.4 and 0.1% at pH 6.0. Compared to the large change of the uncharged fraction (one twenty-sixth at pH 6.0 and eightfold at pH 8.4 compared with pH 7.4), the acidification (pH 6.0) or alkalization (pH 8.4) caused a small change in diphenhydramine uptake (two-fifths at pH6.0 and twofold at pH 8.4 of control uptake at pH7.4), suggesting that passive diffusion according to the pH-partition theory could not be solely responsible for diphenhydramine uptake by hCMEC/D3 cells. This view is further supported by the result that an outward proton gradient from intracellular fluid to extracellular medium effectively enhanced diphenhydramine uptake by hCMEC/D3 cells.
The results of the inhibition study (Figure 4 and Table 3) also indicate that H+/OC antiporter is functionally expressed in hCMEC/D3 cells. Diphenhydramine and pyrilamine each mutually inhibited the uptake of the other, suggesting the occurrence of competition between oxycodone and pyrilamine for a common transporter. Oxycodone also competitively inhibited diphenhydramine transport in hCMEC/D3 cells (Figure 4). A variety of organic cations with widely differing molecular structures (type II cations), such as pyrilamine, oxycodone, quinidine and amantadine, markedly inhibited diphenhydramine uptake by hCMEC/D3 cells (Figure 4 and Table 3). These results are consistent with those obtained in TR-BBB13 cells [9, 10, 20]. In contrast, TEA and serotonin, which are prototypical substrates/inhibitors of OCT1-3 and PMAT, respectively, had no significant effect. Given that the transport activities of OCT1-3 and PMAT are reduced by membrane depolarization, it is unlikely that these cation transporters are the molecular entity of the H+/OC antiporter. Low or negligible expression of hOCT1-2 mRNA in hCMEC/D3 cells also supports this idea (Table 4).
Quantitative RT-PCR analysis showed that the expression level of hOCTN2 was the highest in hCMEC/D3 cells, followed by hOCTN1, hPMAT, hOCT3 and hOCT1. On the other hand, the expression levels of hOCT2 and hMATE1-2 were negligible or low in hCMEC/D3 cells. Expression levels of these mRNAs in hCMEC/D3 cells were similar to those reported in TR-BBB13 cells  and rat brain capillary endothelial cells (RBEC1) . The H+/OC antiporter at the BBB remains molecularly unidentified even in rodents. Because hCMEC/D3 cells possess a H+/OC antiporter, like rodent BBB, and show similar mRNA expression of identified organic cation transporters to those in rat BBB model cells, hCMEC/D3 cells should be a good in vitro model for further studies on the H+/OC antiporter. Kooijmans et al. have reported that amino acid transporter B0,+ (SLC6A14) is involved in Na+- and Cl--dependent amantadine transport in hCMEC/D3 cells . Although amantadine inhibited diphenhydramine transport in hCMEC/D3 cells, diphenhydramine transport was insensitive to extracellular Na+. Thus, an unidentified transport system different from SLC6A14 seems to be a candidate for the H+/OC antiporter.
An aim of this study was to investigate whether or not the results of in vitro uptake study using hCMEC/D3 cells can be extrapolated to the human BBB. As a first step, the influx BBB permeability-surface area product (PSBBB,inf) for diphenhydramine and 3H]pyrilamine in rats were compared with those measured by the in vitro uptake study using TR-BBB13 cells. The value of PSBBB,inf measured by the in situ brain perfusion is mainly reflected in the unidirectional clearance from perfusate to brain across the BBB, as far as the BBB transport process is the rate-limiting step . The values of PSBBB,inf was estimated to be 44 and 13 μL/min/cm2 for diphenhydramine and 3H]pyrilamine, respectively, assuming that the rat brain capillary surface area is 100 cm2/g of brain . These values approximate to the in vitro uptake clearances in TR-BBB13 cells for diphenhydramine (21 μL/min/cm2)  and 3H]pyrilamine (6.3 μL/min/cm2) . These results indicate the possibility that the in vivo BBB permeability can be roughly predicted from the in vitro uptake clearance estimated by BBB model cells, as far as diphenhydramine and pyrilamine. In hCMEC/D3 cells, in vitro uptake clearance for 3H]pyrilamine is estimated to be 8.39 μL/min/cm2, which is in fairly good agreement with the in vivo human BBB of 11C]pyrilamine (15 μL/min/cm2) estimated from a positron emission tomography (PET) study . Extensive further studies and human data will be needed to allow reliable prediction of human BBB permeability from in vitro uptake studies using hCMEC/D3 cells.
Our results strongly suggest that H+/OC antiporter is functionally expressed in the immortalized human brain capillary endothelial cell line hCMEC/D3. Like the putative H+/OC antiporter in rodents, the transporter was energy-dependent and also dependent on an oppositely directed proton gradient, but was sodium ion- or membrane potential-independent. These findings should be relevant to the development and clinical application of CNS-acting cationic drugs in humans. We suggest that the hCMEC/D3 cell line should be a useful model system in the development of new CNS-acting drugs and optimal pharmacotherapy for various CNS diseases.
Proton-coupled organic cation
Plasma membrane monoamine transporter
Central nervous system
Brain interstitial fluid
The authors would like to thank Prof Margareta Hammarlund-Udenaes (Department of Pharmaceutical Bioscience, Uppsala University, Uppsala, Sweden) for helpful discussion. We also would like to thank Prof. Sumio Ohtsuki (Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan) for mediating the supply of hCMEC/D3 cells. This work was supported in part by a Grant-in-Aid for Scientific Research provided by the Ministry of Education, Culture, Sports, Science and Technology, and also by the Japan Society for the Promotion of Science (JSPS) and Centre National de la Recherche Scientifique (CNRS) under the Japan-France Basic Scientific Cooperation Program.
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