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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.

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.
Keita Shimomura, Takashi Okura, Sayaka Kato, Pierre-Olivier Couraud, Jean-Michel Schermann, Tetsuya Terasaki, Yoshiharu Deguchi To cite this version: Keita Shimomura, Takashi Okura, Sayaka Kato, Pierre-Olivier Couraud, Jean-Michel Scher-mann, et al. Functional expression of a proton-coupled organic cation (H+/OC) antiporter inhuman brain capillary endothelial cell line hCMEC/D3, a human blood-brain barrier model.
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publics ou priv´ Shimomura et al. Fluids and Barriers of the CNS 2013, 10:8 FLUIDS AND BARRIERS Functional expression of a proton-coupledorganic cation (H+/OC) antiporter in human braincapillary endothelial cell line hCMEC/D3, a humanblood–brain barrier modelKeita Shimomura1, Takashi Okura1, Sayaka Kato1, Pierre-Olivier Couraud2,3,4, Jean-Michel Schermann5,6,Tetsuya Terasaki7 and Yoshiharu Deguchi1* Background: Knowledge of the molecular basis and transport function of the human blood–brain barrier (BBB) isimportant for not only understanding human cerebral physiology, but also development of new central nervoussystem (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 functionalexpression of a proton-coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell linehCMEC/D3, which has been recently developed as an in vitro human BBB model.
Methods: Diphenhydramine, [3H]pyrilamine and oxycodone were used as cationic drugs that proved to be H+/OCantiporter substrates. The in vitro uptake experiments by hCMEC/D3 cells were carried out under several conditions.
Results: Diphenhydramine and [3H]pyrilamine were both transported into hCMEC/D3 cells in a time- andconcentration-dependent manner with Km values of 59 μM and 19 μM, respectively. Each inhibited uptake of theother in a competitive manner, suggesting that a common mechanism is involved in their transport. Thediphenhydramine uptake was significantly inhibited by amantadine and quinidine, but not tetraethylammoniumand 1-methyl-4-phenylpyridinium (substrates for well-known organic cation transporters). The uptake was inhibitedby metabolic inhibitors, but was insensitive to extracellular sodium and membrane potential. Further, the uptakewas increased by extracellular alkalization and intracellular acidification. These transport properties are completelyconsistent with those of previously characterized H+/OC antiporter in rat BBB.
Conclusions: The present results suggest that H+/OC antiporter is functionally expressed in hCMEC/D3 cells.
Keywords: Human blood–brain barrier, Human BBB model cell, hCMEC/D3 cells, Proton-coupled organic cationantiporter, Organic cation transporter, Transport function, Diphenhydramine, Pyrilamine, Oxycodone, Activetransport, Real-time PCR * Correspondence: 1Department of Drug Disposition and Pharmacokinetics, School ofPharmaceutical Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo173-8605, JapanFull list of author information is available at the end of the article 2013 Shimomura et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.
Shimomura et al. Fluids and Barriers of the CNS 2013, 10:8 substrates are secondary or tertiary amines with positive The human brain is protected by the existence of the charge at physiological pH. This suggests that many CNS blood–brain barrier (BBB), which consists of brain capil- drugs used in the clinical setting may be efficiently taken lary endothelial cells linked with tight junctions It is up into the brain via the H+/OC antiporter at the BBB. In well established that the polarized expression of numer- addition, this putative transporter is a potential target in the ous transporters and receptors at the brain capillary development of new CNS drugs.
endothelial cells controls the blood–brain exchange of The purpose of this study, therefore, is to clarify nutrients, waste products produced from neurotransmit- the functional expression of the H+/OC antiporter in ter substances, and drugs Therefore, knowledge of the hCMEC/D3 cells. We also discuss whether or not the molecular basis and transport function of the human BBB results of in vitro uptake study using hCMEC/D3 cells is important for not only understanding human cerebral can be extrapolated to the human BBB in vivo, as well physiology, but also for development of new central ner- as the relevance of our findings to cerebral physiology vous system (CNS)-acting drugs. However, few studies and to the development and proper use of CNS-acting have been done using human brain capillary endothelial cationic drugs.
cells, because human brain materials are difficult to ob-tain. In addition, isolation and primary culture of brain ca- pillary endothelial cells are laborious and time-consuming procedures [. Therefore, the development of simple Diphenhydramine hydrochloride was purchased from in vitro BBB models is highly desirable.
Wako Pure Chemical Industries (Osaka, Japan), [3H]Pyrila- Human immortalized brain capillary endothelial cells mine (23 – 30 Ci/mmol) and [14C]inulin (2.06 mCi/g) were (hCMEC/D3) have recently been developed as an in vitro purchased from Amersham Bioscience (Buckingshamshire, human BBB model [This cell line has been now exten- UK) and PerkinElmer Life and Analytical Sciences, Inc.
sively validated by numerous laboratories worldwide in (Walthan, Massachusetts, USA), respectively. Oxycodone pharmacological, toxicological, immunological and infec- was kindly provided by Takeda Pharmaceutical Co. Ltd.
tion studies. These hCMEC/D3 cells retain many of the (Osaka, Japan). All other chemicals and reagents, in- morphological and functional characteristics of the human cluding diphenhydramine, were commercial products of BBB in terms of expression of multiple transporters, recep- reagent grade.
tors, tight junction proteins and various ABC transporters,including ABCB1 (MDR1/P-gp), ABCC1 (MRP1), ABCC4 (MRP4), ABCC5 (MRP5), and ABCG2 (BCRP) Fur- The hCMEC/D3 cells had been immortalized by lentiviral thermore, several solute carrier (SLC) transporters respon- transduction of the catalytic subunit of human telomerase sible for the blood–brain exchange of mainly nutrients, and SV40-T antigen . The cells were cultivated at 37°C including SLC2A1 (GLUT1), SLC16A1 (MCT1), SLC29A1 in EBM-2 medium (Takara Bio, Shiga, Japan) supplemen- (ENT1) and so on, are highly expressed at the mRNA level ted with 2.5% fetal bovine serum, 0.025% VEGF, 0.025% in this cell line [On the other hand, little is known R3-IGF, 0.025% hEGF, 0.01% hydrocortisone, 5 μg/mL concerning the expression and function of influx transpor- bFGF, 1% penicillin-streptomycin and 10 mM HEPES on ters that may regulate the brain distribution of drugs, rat collagen type I coated dishes in 95% air and 5% CO2.
except for relatively abundant expression of SLCO2A1(OATP2A1) at the mRNA level Recently, we have reported that a H+/OC antiporter is Adult male Wistar rats weighing about 350 g were pur- functionally expressed in the in vivo rat BBB and also in chased from Japan SLC (Shizuoka, Japan); they were a conditionally immortalized rat BBB cell line (TR-BBB13 housed, three or four per cage, in a laboratory with free cells) [This H+/OC antiporter mediates blood–brain access to food and water and were maintained on a transport of CNS-acting cationic drugs such as pramipex- 12-hr dark/12-hr light cycle in a room with controlled ole, oxycodone and diphenhydramine, in addition to pyrila- temperature (24 ± 2°C) and humidity (55 ± 5%). This mine, in rats. A brain microdialysis study revealed that this study was conducted according to guidelines approved transporter actively transports oxycodone and diphen- by the Experimental Animal Ethical Committee of hydramine into the brain, and their unbound concentration Teikyo University.
in brain interstitial fluid (ISF) is 3- to 5-fold higher thanthat in blood There is also evidence that clonidine Transport studies and methylenedioxymethamphetamine (MDMA) The hCMEC/D3 cells used for the experiments were be- are transported by H+/OC antiporter in the BBB and in tween passage 25 and 35. The cells were seeded on rat peripheral cell lines, respectively. Although the molecular collagen I-coated multi-well plates (Becton Dickinson) at entity of this transporter remains unknown, the known a density of 0.2 × 105 cells/cm2. At 3 days after seeding Shimomura et al. Fluids and Barriers of the CNS 2013, 10:8 the cells reached confluence, the cells were washed twice 25 μM rotenone (dissolved in the transport medium with 2 mL of phosphate-buffered saline (pH 7.4) and containing 0.25% ethanol) or 0.1% NaN3 for 20 min. In this preincubated with the transport buffer (122 mM NaCl, 3 experiment, 10 mM D-glucose in the transport medium mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 1.4 mM was replaced with 10 mM 3-O-methylglucose to reduce CaCl2, 10 mM D-glucose, 10 mM HEPES, pH 7.4) for 20 metabolic energy. In order to examine the sodium require- min at 37°C. After preincubation, 1 mL of the transport ment of diphenhydramine uptake by hCMEC/D3 cells, buffer containing diphenhydramine (30 μM) or [3H]pyri- sodium ions were replaced with N-methylglucamine+. To lamine (74 kBq/μL, 90 nM) was added to initiate uptake.
examine the effects of reducing the membrane potential The cells were incubated at 37°C for a designated time, and proton gradient on diphenhydramine uptake by and then washed three times with 2 mL of ice-cold incu- hCMEC/D3 cells, 10 mM valinomycin and 10 μM carbonyl bation buffer to terminate the uptake. In the case of di- cyanide p-trifluoromethoxyphenylhydrazone (FCCP, a pro- phenhydramine, the cells were collected in 400 μL of tonophore) (dissolved in the transport medium containing 0.5% KH2PO4 solution, and stored at −20°C until HPLC 0.22% ethanol), respectively, were added to the transport determination as described below. For the determination medium. These studies were performed in parallel with of [3H]pyrilamine radioactivity, the cells were solubilized controls in the presence of the corresponding ethanol con- with 1 M NaOH for 60 min and the radioactivity was centration. Uptake was also measured at medium pH measured using a liquid scintillation counter after the values of 6.0, 7.4 and 8.4. When the influence of intracellu- addition of scintillation cocktail Hionic Fluor (PerkinElmer lar pH (pHi) was examined, the uptake was measured in Life and Analytical Sciences). Cellular protein content was the presence of 30 mM NH4Cl to elevate pHi []. To determined with a BCA protein assay kit (Pierce Chemical measure the uptake at acidic pHi, extracellular NH4Cl was Co., Rochford, IL, USA).
removed after the preincubation with 30 mM NH4Cl, be- Uptake was expressed as the cell-to-medium ratio (μL/ cause intracellular NH3 rapidly diffuses out of the cells, mg protein) obtained by dividing the uptake amount by resulting in the accumulation of protons released from the concentration of substrate in the transport buffer. In NH+4 during NH3 generation in the cells. In the inhibition order to estimate transporter-mediated specific uptake study, the uptake was measured after incubation with di- parameters in the kinetic analyses, the cell-to-medium phenhydramine (30 μM) for 15 sec in the presence of ratios for saturable components of diphenhydramine (30 – selected inhibitors (amantadine, quinidine, TEA, serotonin, 500 μM, for 15 sec) and [3H]pyrilamine (6 – 500 μM, for MPP+ and choline) at the concentration of 1 mM. Diphen- 10 sec) were determined by subtracting the cell-to-medium hydramine uptake (30 – 500 μM, 15 sec) was measured in ratios at the concentration of 5 mM diphenhydramine and the absence and presence of pyrilamine (150 μM) or oxy- 5 mM pyrilamine, respectively. The non-saturable uptake codone (500 μM). [3H]pyrilamine uptake (6 – 500 μM, 10 component can be estimated from the uptake in the pres- sec) was also measured in the absence and presence of di- ence of an excess of substrate. The initial uptake rates for phenhydramine (50 μM).
saturable components were determined by multiplying thecell-to-medium ratios for saturable component by the sub- In situ brain perfusion study strate concentration in the transport buffer The data Brain perfusion was performed by the same method as were fitted to the following equation to estimate the kinetic reported previously In brief, each rat was anesthe- parameters by means of nonlinear least-squares regression tized and the right carotid artery was catheterized with analysis with Prism software (Graphpad, San Diego, CA, polyethylene tubing (SP-10) filled with sodium heparin (100 IU/mL). The perfusate (Krebs-Henseleit buffer, 118 mMNaCl, 4.7 mM KCl, 25 mM NaHCO3, 1.2 mM KH2PO4, 2, 1.2 mM MgSO4, 10 mM D-glucose, pH 7.4) containing diphenhydramine (10 μM) or [3H]pyrilamineand [14C]inulin (0.9 μM), a brain intravascular marker, was where V is the initial uptake rate of substrate (nmol/min/ passed through the catheter at the rate of 4.9 mL/min with mg protein), s is the substrate concentration in the medium an infusion pump (Harvard Apparatus, South Natick, MA, (μM), Km is the Michaelis-Menten constant (μM) and Vmax USA). After the infusion pump is started, 5.0 sec is required is the maximum uptake rate (nmol/min/mg protein). Vmax/ to fill the external carotid artery cannula Therefore, Km (pmol/min/mg protein/μM = μL/min/mg protein) 5.0 sec was routinely subtracted from the gross perfusion values were calculated as the uptake clearance for the satur- time in each experiment, to obtain the uptake time for able transport component.
which the perfusate was actually within the brain capillaries.
In order to examine the energy dependency of diphen- At the end of uptake for 5 – 30 sec, rats were decapitated, hydramine uptake by hCMEC/D3 cells, the uptake was and the right cerebral hemisphere was dissected from the measured as described above after pretreatment with perfused brain and weighed. The brain samples were stored Shimomura et al. Fluids and Barriers of the CNS 2013, 10:8 at −20°C until determination of diphenhydramine. The hCMEC/D3 cells were measured by quantitative real-time brain uptake of [3H]pyrilamine (1.1 nM) was also deter- PCR analysis. Total RNA was isolated from hCMEC/D3 mined as described above. The radioactivity was measured cells using an RNeasy mini kit (Qiagen, Valencia, CA, using a liquid scintillation counter as described above, USA). Single-strand cDNA was prepared from 1.0 μg of after solubilization of the cerebral hemisphere in Soluene- total RNA by RT (Superscript III, Invitrogen, Carlsbad, 350 (PerkinElmer Life and Analytical Sciences, Boston, CA, USA) using random primers. Quantitative real-time MA, USA) at 55°C for 3 h and decolorization by addition PCR analysis was performed using a 7500 sequence detec- of 0.3 mL of 30% H2O2. The value of the permeability- tion system (PE Applied Biosystems, Foster City, CA, surface area product (PSBBB,inf), which represents in vivo USA) with 2x SYBR Green PCR Master Mix (PE Applied BBB permeability, was calculated after correcting for Biosystems) according to the manufacturer's protocols.
remaining intravascular diphenhydramine or [3H]pyrila- The primers are listed in Table The thermal protocol mine, estimated from the apparent brain uptake of [14C] was set to 2 min at 50°C, followed by 10 min at 95°C, and then 40 cycles of 15 sec at 95°C and 1 min at 60°C. Therelative expression levels of these mRNAs were calculated Determination of diphenhydramine using the comparative Ct method for relative quantifica- The collected cells in 0.5% KH2PO4 solution were homoge- tion based on the mRNA level of the housekeeping gene nized by sonication. For brain tissue, the cerebral hemi- encoding GAPDH, according to the manufacturer's proto- sphere was homogenized in 5 volumes of 0.5% KH2PO4 cols. The control, lacking the RT enzyme, was assayed in solution. To the homogenate (300 μL) was added orphena- parallel to monitor genomic contamination. To confirm drine hydrochloride (50 μM, 6.3 μL) as an internal stand- specificity of amplification, the PCR products were sub- ard, as well as 60 μL of saturated K2CO3. After mixing, jected to melting curve analysis.
samples were extracted with 1.5 mL of hexane-isopropanol(98:2, v/v) by shaking on a horizontal shaker for 15 min Statistical analysis and then centrifuged for 10 min at 800 g. The upper or- Statistical analysis of the data was performed by employ- ganic layer was transferred into a tube containing 300 μL of ing Student's t-test and by one-way analysis of variance 0.5% KH2PO4. The tube was shaken for 15 min and centri- followed by Dunnett's test for single and multiple com- fuged for 10 min at 800 g. The upper organic layer was parisons, respectively. Differences were considered sta- aspirated off, then 60 μL of saturated K2CO3 was added to tistically significant at P < 0.05.
the lower aqueous phase and the extraction step and back-extraction step were repeated. The final K2CO3 solution Table 1 Sense and antisense primers for quantitative PCR 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 (Accession number) Diphenhydramine was determined by ultra-performance liquid chromatography (UPLCW) with a UV detector by modification of previously reported methods ]. A 7.5 μL aliquot was injected into the UPLCW. The UPLCW system (Waters ACQUITY, Milford, MA, USA) consisted of a binary solvent manager, sample manager and UV de-tector. 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 diphen- hydramine and orphenadrine were 6.4 and 9.1 min,respectively. The detection limit for quantification of di- phenhydramine was 75 pmol.
Expression profiling of organic cation transporters by The mRNA levels of typical organic cation transporters (hOCT1-3, hOCTN1-2, hMATE1-2, hPMAT) in the Shimomura et al. Fluids and Barriers of the CNS 2013, 10:8 ResultsUptake kinetics of diphenhydramine and [3H]pyrilamine by hCMEC/D3 cellsThe uptake of diphenhydramine (30 μM) increased in proportion with time until 60 sec at 37°C, and reached equilibrium with the cell-to-medium (C/M) ratio of 97.7 –103 μL/mg protein at 60 – 180 sec (Figure ). [3H]Pyrila- um (C/M) ratio
mine uptake (74 kBq/μL, 90 nM) by hCMEC/D3 cells also L/mg protein
increased linearly with time until 30 sec, and the C/M ratio was 161 – 186 μL/mg protein at 30 – 60 sec (Figure ). Therefore, the initial uptake rate was assessed at 15 sec for diphenhydramine and 10 sec for [3H]pyrila- mine, in the subsequent kinetic and inhibition studies.
The initial uptakes of diphenhydramine and [3H]pyrila- Time (sec)
mine were concentration-dependent (Figures B and ). Eadie-Hofstee plot for diphenhydramine uptakegave a single straight line (r2 = 0.859), indicating in- volvement of a single saturable process (Figure C).
Although the Eadie-Hofstee plot for [3H]pyrilamine uptake has the lower r2 value (0.679) than that for di- phenhydramine, the kinetic parameters for both drugs were analyzed assuming that each drug has a singlesaturable transport process, on the basis of our previous Initial uptake rate
reports [Kinetic analysis provided a Km value of V (nmol/mg
59 μM and a Vmax of 13 nmol/mg protein/min for di- phenhydramine, and a Km value of 19 μM and a Vmax of 3.5 nmol/mg protein/min for [3H]pyrilamine. The Vmax/ Diphenhydramine concentration (µ M)
Km values (uptake clearance) for diphenhydramine and[3H]pyrilamine were 220 and 184 ( μL/min/mg protein), Metabolic energy and ion dependence of the uptake of diphenhydramine by hCMEC/D3 cellsDiphenhydramine uptake in hCMEC/D3 cells was sig- nificantly inhibited by pretreatment with rotenone and sodium azide, but was not affected by replace-ment of extracellular sodium ion with N-methylgluca- mine+ or by treatment with valinomycin, a potassium ionophore (Table ).
V / [S] (mL/mg protein/15 sec)
The C/M ratio of diphenhydramine was decreased in Figure 1 Diphenhydramine uptake by hCMEC/D3 cells. (A) Time acidic transport medium (pH 6.0) and significantly course of the cell-to-medium (C/M) ratio of diphenhydramine increased in alkaline medium (pH 8.4) by 2.1-fold, com- uptake by hCMEC/D3 cells. Uptake of diphenhydramine (30 μM) was pared with that at pH 7.4 (Figure ; it was also measured at 37°C. (B) Concentration-dependence of the initial decreased by treatment with FCCP, a protonophore uptake rate of diphenhydramine in hCMEC/D3 cells. hCMEC/D3 cellswere preincubated with incubation buffer (122 mM NaCl, 3 mM KCl, (Table To examine the effect of protons as a driving 25 mM NaHCO3, 1.2 mM MgSO4, 1.4 mM CaCl2, 10 mM D-glucose, force, the cells were treated with NH4Cl, because the 10 mM HEPES, pH 7.4) for 20 min at 37°C. Uptake of intracellular pH (pHi) value rises in the presence of diphenhydramine (30 μM) was measured at 37°C for 15 sec. (C) Eadie-Hofstee plot of diphenhydramine uptake by hCMEC/D3 cells.
4Cl (acute treatment), whereas pretreatment and subsequent removal of NH V, initial uptake rate (nmol/mg protein/15 sec). [S], diphenhydramine 4Cl (pretreatment) causes a concentration in the transport medium ( decrease in the pHi value. Intracellular alkalization μM). Each point represents the mean ± S.E. from three to four determinations. When verticals markedly reduced diphenhydramine uptake, while intra- are not shown, the S.E. is contained within the limits of the symbols.
cellular acidification resulted in stimulation of diphen-hydramine uptake (Figure ).
Shimomura et al. Fluids and Barriers of the CNS 2013, 10:8 Table 2 Effects of metabolic inhibitors (rotenone and sodium azide), protonophore (FCCP), sodium replacement and change in membrane potential byvalinomycin on diphenhydramine uptake by Relative uptake (% of control) um (C/M) ratio
Rotenone (25 μM) a), b) L/mg protein
Sodium azide (0.1%) b) Valinomycin (10 μM) c) Time (sec)
Uptake of diphenhydramine (30 μM) was measured at 37°C for 15 sec in theabsence or presence of metabolic inhibitors (25 μM rotenone and 0.1% sodium azide) and a protonophore (10 μM p- FCCP) and 10 µM valinomycin.
The uptake was also measured in sodium-free incubation buffer (Na+ was replaced with N-methylglucamine). a) Rotenone was dissolved in the transport buffer containing 0.25% ethanol. b) 25 µM rotenone and 0.1% sodium azide were added to the transport buffer without 10 mM D-glucose to reduce metabolic energy. c) The cells were preincubated with the transport buffer containing 10 μM valinomycin for 10 min. Valinomycin was dissolved in the transport buffer containing 0.22% ethanol. Each study was performed in parallel with a control containing the corresponding ethanol concentration.
Each value represents the mean as % of control ± S.E. (n = 3). **p<0.01 and **p<0.001 vs control.
Initial u
Inhibition of uptake of diphenhydramine and [3H] pyrilamine by hCMEC/D3 cells In Lineweaver-Burk plot analyses of mutual inhibitory [3H]Pyrilamine concentration (µ M)
effects on uptake of diphenhydramine and pyrilamine (Figure , the plots of diphenhydramine uptake in the presence and absence of pyrilamine intersected at the ordinate axis (Figure This result indicated that pyrila- mine competitively inhibited diphenhydramine uptake witha Ki value of 240 μM. Diphenhydramine also competitively inhibited [3H]pyrilamine uptake with a Ki value of 28.9 μM(Figure B). These mutual and competitive inhibitions between diphenhydramine and pyrilamine suggest that a common transporter is involved in their influx transport into the hCMEC/D3 cells, at least in part. Further, diphen- hydramine uptake was competitively inhibited by oxy- V / [S] (mL/mg protein/10 sec)
codone with a Ki value of 304 μM (Figure In the Figure 2 [3H]pyrilamine uptake by hCMEC/D3 cells. (A) Time inhibition study, diphenhydramine uptake was significantly course of the cell-to-medium (C/M) ratio of [3H]pyrilamine uptake by inhibited by the cationic compounds amantadine and hCMEC/D3 cells. Uptake of [3H]pyrilamine (74 kBq/μL, 90 nM) was quinidine to less than 25% of the control, while TEA measured at 37°C. (B) Concentration-dependence of the initialuptake rate of [3H]pyrilamine in hCMEC/D3 cells. hCMEC/D3 cells (a classical substrate and/or inhibitor of OCTs), MPP+ were preincubated with incubation buffer (122 mM NaCl, 3 mM KCl, (a classical substrate and/or inhibitor of PMAT as well as 25 mM NaHCO3, 1.2 mM MgSO4, 1.4 mM CaCl2, 10 mM D-glucose, OCTs), serotonin, and choline (a substrate or inhibitor of 10 mM HEPES, pH 7.4) for 20 min at 37°C. Uptake of [3H]pyrilamine the choline transport system) had little effect (Table (74 kBq/μL, 90 nM) was measured at 37°C for 10 sec. (C) Eadie-Hofstee plot of [3H]pyrilamine uptake by hCMEC/D3 cells. V, initial mRNA expression of organic cation transporters in uptake rate (nmol/mg protein/10 sec). [S], [3H]pyrilamineconcentration in the transport medium (μM). Each point represents the mean ± S.E. from three to four determinations. When verticals To provide molecular evidence for the expression of or- are not shown, the S.E. is contained within the limits of the symbols.
ganic cation transporters in hCMEC/D3 cells, the mRNAexpression levels of hOCT1, hOCT2, hOCT3, hOCTN1,hOCTN2, hMATE1, hMATE2 and hPMAT in hCMEC/D3 cells were determined by quantitative RT-PCR analysis(Table . Relatively high mRNA expression of hOCTN2 Shimomura et al. Fluids and Barriers of the CNS 2013, 10:8 + pyrilamine
of control)
(nmol/mg protein 15 sec)
1/S ( µ M)
(nmol/mg protein 10 sec)
of control)

1/S ( µ M)
NH Cl (acute) NH Cl (pre)
+ oxycodone
Figure 3 Effect of extracellular pH and intracellular pH ondiphenhydramine uptake by hCMEC/D3 cells. (A) Uptake of diphenhydramine (30 μM) was measured at 37°C for 15 sec atmedium pH values of 6.0, 7.4 and 8.4. Each column represents the mean ± S.E. of three determinations. Asterisks show a significant (nmol/mg protein 15 sec)
difference, *P<0.05 vs pH 7.4 (control). (B) Uptake of diphenhydramine (30 μM) was measured at 37°C, pH 7.4 for 15 sec.
The uptake was measured in the presence of 30 mM NH4Cl to elevate intracellular pH (pHi) (NH4Cl (acute)). To measure the uptake 1/S ( µ M)
at acidic pHi, extracellular NH4Cl was removed after preincubationwith 30 mM NH Figure 4 Lineweaver-Burk plot of mutual inhibitory effects on 4Cl for 20 min (NH4Cl (pre)). Each column represents the mean ± S.E. of three determinations. Asterisks show a significant uptakes of diphenhydramine or [3H]pyrilamine by hCMEC/D3 difference, **P<0.01 vs control.
cells. Diphenhydramine uptake was measured at 37°C for 15 sec inthe presence () or absence (○) of pyrilamine (150 μM) (A) oroxycodone (500 μM) (C). [3H]Pyrilamine uptake was also measured was found, followed by hOCTN1 and hPMAT. In at 37°C for 10 sec in the presence () or absence (○) of contrast, little expression of hOCTs and hMATEs mRNAs diphenhydramine (50 μM) (B). Each point represents the mean ± S.E.
was seen in hCMEC/D3 cells.
of four determinations.
In vivo blood-to-brain transport of diphenhydramine and [3H]pyrilamine were 4.40 and 1.30 mL/min/g brain, respect- [3H]pyrilamine in rats ively. The PSBBB,inf value for [3H]pyrilamine was similar to The brain uptakes of diphenhydramine and [3H]pyrilamine the value reported previously (1.6 mL/min/g brain) were measured by the in situ brain perfusion technique. Thebrain/perfusate (B/P) ratios of diphenhydramine and [3H] pyrilamine linearly increased with increasing perfusion time There is increasing evidence that the immortalized up to 30 sec. The PSBBB,inf values for diphenhydramine and human brain capillary endothelial cell line hCMEC/D3 is Shimomura et al. Fluids and Barriers of the CNS 2013, 10:8 Table 3 Inhibitory effects of various compounds on present study, diphenhydramine and pyrilamine were used diphenhydramine uptake by hCMEC/D3 cells as substrates to investigate this activity.
Concentration (mM) Relative uptake (% of control) The uptakes of diphenhydramine and [3H]pyrilamine were time- and concentration-dependent. Kinetic analysesrevealed 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 agree- ment with those of diphenhydramine (440 μL/min/mg protein) and [3H]pyrilamine (140 μL/min/mg protein) in conditionally immortalized rat brain capillary endothelial The inhibitory effects of selected compounds on diphenhydramine uptake by cells (TR-BBB13) These results suggest that a trans- hCMEC/D3 cells. Uptake of diphenhydramine (30 μM) was measured at 37°C porter with similar transport activity and transport clear- for 15 sec in the transport medium (pH 7.4) containing each compound(1 mM). Each column represents mean ± S.E. of three determinations.
ance to those observed in the case of TR-BBB13 cells is Asterisks show a significant difference, *** P < 0.01 vs control.
involved in uptake of diphenhydramine and [3H]pyrila-mine by hCMEC/D3 cells. Furthermore, the transporter a good model to predict BBB permeability in the human seems to show no marked species difference between brain In the present study, we further investigated TR-BBB13 cells and hCMEC/D3 cells.
the utility of this model by examining whether H+/OC The uptake of diphenhydramine was significantly inhib- ited by pretreatment with metabolic inhibitors, but was in- Although this putative H+/OC antiporter still remains to sensitive to extracellular sodium and membrane potential be identified at the molecular level, it is known to trans- in hCMEC/D3 cells (Table suggesting the involvement port several CNS-acting drugs with secondary or tertiary of a transporter having similar energy and membrane po- amine moieties, including diphenhydramine, oxycodone, tential dependencies to those of TR-BBB13 cells. Further- pyrilamine and clonidine more, the diphenhydramine uptake by hCMEC/D3 cells Diphenhydramine, pyrilamine and oxycodone each form showed pH-dependency characteristic of a proton-coupled a cation at physiological pH because they are weak bases antiporter. The uptake was increased at higher extracellular having a tertiary amine moiety. We previously showed pH (pH 8.4), and decreased in the presence of FCCP. Intra- that diphenhydramine, pyrilamine and oxycodone are cellular acidification induced with NH4Cl, stimulated the taken up via a pH-sensitive, energy-dependent, proton- uptake (Figure and Table ). As the pKa value of diphen- coupled antiport system in TR-BBB13 cells, which are an hydramine is 8.98, the proportion of uncharged diphen- in vitro rat BBB model ]. In addition, both diphen- hydramine can be estimated to be 20.8% at pH 8.4, 2.6% at hydramine and oxycodone have been reported to be pH 7.4 and 0.1% at pH 6.0. Compared to the large change actively taken up by the brain across the BBB with a Kp, of the uncharged fraction (one twenty-sixth at pH 6.0 and uu (unbound concentration ratio of brain interstitial fluid eightfold at pH 8.4 compared with pH 7.4), the acidifica- to plasma) value of more than 3 in rats ]. Conse- tion (pH 6.0) or alkalization (pH 8.4) caused a small change quently, we have suggested that H+/OC antiporter works in diphenhydramine uptake (two-fifths at pH6.0 and two- in the rat BBB as an active influx transporter. In the fold at pH 8.4 of control uptake at pH7.4), suggesting thatpassive diffusion according to the pH-partition theory Table 4 mRNA expression levels of organic cation could not be solely responsible for diphenhydramine up- transporters in hCMEC/D3cells determined by take by hCMEC/D3 cells. This view is further supported by quantitative RT-PCR analysis the result that an outward proton gradient from intracellu- lar fluid to extracellular medium effectively enhanced (target mRNA x 105/GAPDH mRNA) diphenhydramine uptake by hCMEC/D3 cells.
The results of the inhibition study (Figure and Table also indicate that H+/OC antiporter is function- ally 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 ). A variety oforganic cations with widely differing molecular structures (type II cations), such as pyrilamine, oxycodone, quinidine Each value represents the mean ± S.E. of eight determinations. N.D.:not detected.
and amantadine, markedly inhibited diphenhydramine Shimomura et al. Fluids and Barriers of the CNS 2013, 10:8 uptake by hCMEC/D3 cells (Figure and Table These μL/min/cm2, which is in fairly good agreement with the results are consistent with those obtained in TR-BBB13 in vivo human BBB of [11C]pyrilamine (15 μL/min/cm2) cells In contrast, TEA and serotonin, which are estimated from a positron emission tomography (PET) prototypical substrates/inhibitors of OCT1-3 and PMAT, study [Extensive further studies and human data respectively, had no significant effect. Given that the trans- will be needed to allow reliable prediction of human port activities of OCT1-3 and PMAT are reduced by mem- BBB permeability from in vitro uptake studies using brane depolarization, it is unlikely that these cation hCMEC/D3 cells.
transporters are the molecular entity of the H+/OC antipor-ter. Low or negligible expression of hOCT1-2 mRNA in hCMEC/D3 cells also supports this idea (Table Our results strongly suggest that H+/OC antiporter is Quantitative RT-PCR analysis showed that the expres- functionally expressed in the immortalized human brain sion level of hOCTN2 was the highest in hCMEC/D3 capillary endothelial cell line hCMEC/D3. Like the cells, followed by hOCTN1, hPMAT, hOCT3 and hOCT1.
putative H+/OC antiporter in rodents, the transporter On the other hand, the expression levels of hOCT2 and was energy-dependent and also dependent on an oppos- hMATE1-2 were negligible or low in hCMEC/D3 cells.
itely directed proton gradient, but was sodium ion- or Expression levels of these mRNAs in hCMEC/D3 cells membrane potential-independent. These findings should were similar to those reported in TR-BBB13 cells [and be relevant to the development and clinical application rat brain capillary endothelial cells (RBEC1) The of CNS-acting cationic drugs in humans. We suggest H+/OC antiporter at the BBB remains molecularly that the hCMEC/D3 cell line should be a useful model unidentified even in rodents. Because hCMEC/D3 cells system in the development of new CNS-acting drugs possess a H+/OC antiporter, like rodent BBB, and show and optimal pharmacotherapy for various CNS diseases.
similar mRNA expression of identified organic cationtransporters to those in rat BBB model cells, hCMEC/D3 AbbreviationsBBB: Blood cells should be a good in vitro model for further studies –brain barrier; FCCP: carbonyl cyanide on the H+/OC antiporter. Kooijmans et al. have reported H+/OC: Proton-coupled organic cation; TEA: Tetraethylammonium; that amino acid transporter B0,+ (SLC6A14) is involved in PMAT: Plasma membrane monoamine transporter; CNS: Central nervoussystem; ISF: Brain interstitial fluid; MDMA: Methylenedioxymethamphetamine.
Na+- and Cl--dependent amantadine transport in hCMEC/D3 cells Although amantadine inhibited diphenhydra- Competing interests mine transport in hCMEC/D3 cells, diphenhydramine The authors declare that they have no competing interests.
transport was insensitive to extracellular Na+. Thus, anunidentified transport system different from SLC6A14 Authors' contributionsKS and TO carried out the in vitro transport studies. SK carried out the in vivo seems to be a candidate for the H+/OC antiporter.
animal study. TO carried out the manuscript preparation. PC helped to draft An aim of this study was to investigate whether or not the manuscript, and supplied hCMEC/D3 cells under license from INSERM. JS the results of in vitro uptake study using hCMEC/D3 and TT helped to draft the manuscript. YD supervised the study design andmanuscript preparation. All authors read and approved the final manuscript.
cells can be extrapolated to the human BBB. As a firststep, the influx BBB permeability-surface area product The authors would like to thank Prof Margareta Hammarlund-Udenaes BBB,inf ) for diphenhydramine and [3H]pyrilamine in (Department of Pharmaceutical Bioscience, Uppsala University, Uppsala, rats were compared with those measured by the in vitro Sweden) for helpful discussion. We also would like to thank Prof. Sumio uptake study using TR-BBB13 cells. The value of PSBBB, Ohtsuki (Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan)for mediating the supply of hCMEC/D3 cells. This work was supported in inf measured by the in situ brain perfusion is mainly part by a Grant-in-Aid for Scientific Research provided by the Ministry of reflected in the unidirectional clearance from perfusate to Education, Culture, Sports, Science and Technology, and also by the Japan brain across the BBB, as far as the BBB transport process Society for the Promotion of Science (JSPS) and Centre National de la is the rate-limiting step The values of PS Recherche Scientifique (CNRS) under the Japan-France Basic Scientific estimated to be 44 and 13 μL/min/cm2 for diphenhydra-mine and [3H]pyrilamine, respectively, assuming that the rat brain capillary surface area is 100 cm2/g of brain [.
Department of Drug Disposition and Pharmacokinetics, School of Pharmaceutical Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo These values approximate to the in vitro uptake clearances 173-8605, Japan. 2INSERM, U1016, Institut Cochin, Paris, France. 3CNRS, in TR-BBB13 cells for diphenhydramine (21 μL/min/cm2) UMR8104, Paris, France. 4Université Paris Descartes, Sorbonne Paris Cité, Paris, ] and [3H]pyrilamine (6.3 France. 5Neuropsychopharmacologie des addiction (CNRS UMR 8206), μL/min/cm2) . These Université Paris Decartes, Faculté de Pharmacie, Paris, France. 6INSERM U705, results indicate the possibility that the in vivo BBB perme- Neuropsychopharmacologie des addiction, Paris, France. 7Division of ability can be roughly predicted from the in vitro uptake Membrane Transport and Drug Targeting, Graduate School of clearance estimated by BBB model cells, as far as diphen- Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
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Rapid detection of genetically modified organisms on a continuous-flow polymerase chain reaction microfluidics

Analytical Biochemistry 385 (2009) 42–49 Contents lists available at Analytical Biochemistry Rapid detection of genetically modified organisms on a continuous-flowpolymerase chain reaction microfluidics Yuyuan Li, Da Xing *, Chunsun Zhang MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, No. 55, Zhongshan Avenue West, Tianhe District,Guangzhou 510631, People's Republic of China

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