Characterization of uptake of folates by rat and human blood-brain barrier endothelial cells.
ABSTRACT This study aimed to characterize (3)H-folic acid ((3)H-FA) and (14)C-methyltetrahydrofolic acid ((14)C-MTHF) uptake by rat (RBE4) and human (hCMEC/D3) blood-brain barrier (BBB) endothelial cell lines. Uptake of (3)H-FA and (14)C-MTHF by RBE4 cells was time dependent and linear for the first 2 min of incubation; uptake by hCMEC/D3 cells showed a less marked time-dependency and a greater experimental variability. So, further experiments were performed in RBE4 cells only. Uptake of (3)H-FA was stimulated at acidic and alkaline pH, Na(+) dependent, stimulated when F(-) substituted for Cl(-), energy independent, inhibited by premetrexed, stimulated by cytochalasin D, and unaffected by MTHF, DIDS, SITS, methotrexate, monensin, and FA. Uptake of (14)C-MTHF was found to be pH-, Na(+)-, Cl(-)- and energy independent, inhibited by premetrexed and methotrexate, stimulated by cytochalasin D, and unaffected by FA, DIDS, SITS, monensin, and MTHF. RT-PCR analysis showed mRNA expression of reduced folate transporter (RFC), but neither of FRalpha nor of proton-coupled folate transporter (PCFT) in RBE4 cells, and mRNA expression of RFC and PCFT, but not of FRalpha, in hCMEC/D3 cells. In conclusion, both human and rat BBB endothelial cells show little capacity for (3)H-FA and (14)C-MTHF apical uptake. Hence, these cell lines do not appear to be a good model to study the transport of folates at the BBB.
- SourceAvailable from: vt.edu[show abstract] [hide abstract]
ABSTRACT: (1) In vitro models of the BBB have been developed from cocultures between bovine, porcine, rodent or human brain capillary endothelial cells with rodent or human astrocytes. Since most in vivo BBB studies have been performed with small laboratory animals, especially rats, it is important to establish a rat brain endothelial (RBE) cell culture system that will allow correlations between in vitro and in vivo results. The present review will constitute a brief description of the best characterized RBE cell lines (RBE4, GP8/3.9, GPNT, RBEC1, TR-BBBs and rBCEC4 cell lines) and will summarize their recent and important contribution to our current knowledge of the BBB transport functions and permeability to blood-borne solutes, drugs, and cells. (2) In most cases, primary cultures of RBE cells were transduced with an immortalizing gene (SV40 or polyoma virus large T-antigen or adenovirus E1A), either by transfection of plasmid DNA or by infection using retroviral vectors. In one case however, the conditionally immortalized TR-BBB cell line was derived from primary cultures of brain endothelial cells of SV40-T-expressing transgenic rats. (3) All cell lines appear to have an endothelial morphology. The absence of foci formation would mean that the cells are not transformed. The endothelial origin is shown by the expression of Factor VIII-related antigen. Immortalized RBE cells express all the enzymes and transporters that are considered as specific for the blood-brain barrier endothelium, with similar characteristics to those expected from in vivo analyses, but at a significantly lower level. Some RBE cell lines are responsive to astroglial factors, such as RBE4 cells, rBEC4, and TR-BBB cells. None of the immortalized RBE cell lines appear to generate the necessary restrictive paracellular barrier properties that would allow to use them in transendothelial permeability screening. (4) RBE cell lines have been used to demonstrate that transporters such as organic cation transporter/carnitine transporter, serotonin transporter, and the ATA2 system A isoform are expressed in rat brain endothelium. When the transporter is shown to be expressed with the same properties in the immortalized RBE cells as in vivo, regulation studies may be initiated even if the transporter is down-regulated. Pharmacological applications have been proposed with well-characterized transporters such as monocarboxylic acid transporter-1, large neutral amino acid tansporter-1, nucleoside carrier systems, and P-glycoprotein. RBE cell monolayers have also been used to investigate the mechanism of the transendothelial transport of large molecules, such as immunoliposomes or nanoparticles, potentially useful as drug delivery vectors to the brain. (5) RBE4 and GP8 cell lines have been extensively used to demonstrate that intercellular adhesion molecule-1 (ICAM-1) engagement in brain endothelial cells triggers multiple signal transduction pathways. Using functional assays, it was established that ICAM-1 signaling is intimately and actively involved in facilitating lymphocyte infiltration. (6) Several RBE cell lines have been described, which constitute tentative in vitro models of the rat BBB. The major limitation of these models generally appears to be due to their relatively high paracellular permeability to small molecules, thus limiting their use for permeability studies. The strategies developed for the production of these RBE cell lines will enable the characterization of still more efficient permeability models, as well as the immortalization of human brain endothelial cells.Cellular and Molecular Neurobiology 03/2005; 25(1):41-58. · 2.29 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.The FASEB Journal 12/2005; 19(13):1872-4. · 5.70 Impact Factor
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ABSTRACT: This paper reviews the chemistry, metabolism, and molecular biology of folic acid, with a particular emphasis on how it is, or may be, involved in many disease processes. Folic acid prevents neural tube defects like spina bifida, while its ability to lower homocysteine suggests it might have a positive influence on cardiovascular disease. A role for this B vitamin in maintaining good health may, in fact, extend beyond these clinical conditions to encompass other birth defects, several types of cancer, dementia, affective disorders, Down's syndrome, and serious conditions affecting pregnancy outcome. The effect of folate in these conditions can be explained largely within the context of folate-dependent pathways leading to methionine and nucleotide biosynthesis, and genetic variability resulting from a number of common polymorphisms of folate-dependent enzymes involved in the homocysteine remethylation cycle. Allelic variants of folate genes that have a high frequency in the population, and that may play a role in disease formation include 677C --> T-MTHFR, 1298A --> C-MTHFR, 2756A --> G-MetSyn, and 66A --> G-MSR. Future work will probably uncover further polymorphisms of folate metabolism, and lead to a wider understanding of the interaction between this essential nutrient and the many genes which underpin its enzymatic utilization in a plethora of critical biosynthetic reactions, and which, under adverse nutritional conditions, may promote disease.Molecular Genetics and Metabolism 01/2000; 71(1-2):121-38. · 2.83 Impact Factor
Characterization of uptake of folates
by rat and human blood–brain barrier
Joa ˜o R. Arau ´jo, Pedro Gonc ¸alves, and Fa ´tima Martel*
Department of Biochemistry (U38-FCT), Faculty of Medicine, University of Porto, Porto, Portugal
This study aimed to characterize3H-folic acid (3H-FA) and
14C-methyltetrahydrofolic acid (14C-MTHF) uptake by rat
(RBE4) and human (hCMEC/D3) blood–brain barrier (BBB)
endothelial cell lines. Uptake of3H-FA and14C-MTHF by RBE4
cells was time dependent and linear for the first 2 min of
incubation; uptake by hCMEC/D3 cells showed a less marked
time-dependency and a greater experimental variability. So,
further experiments were performed in RBE4 cells only.
Uptake of3H-FA was stimulated at acidic and alkaline pH, Naþ
dependent, stimulated when F?substituted for Cl?, energy
independent, inhibited by premetrexed, stimulated by
cytochalasin D, and unaffected by MTHF, DIDS, SITS,
methotrexate, monensin, and FA. Uptake of14C-MTHF was
found to be pH-, Naþ-, Cl?- and energy independent, inhibited
by premetrexed and methotrexate, stimulated by cytochalasin
D, and unaffected by FA, DIDS, SITS, monensin, and MTHF. RT-
PCR analysis showed mRNA expression of reduced folate
transporter (RFC), but neither of FRa nor of proton-coupled
folate transporter (PCFT) in RBE4 cells, and mRNA expression
of RFC and PCFT, but not of FRa, in hCMEC/D3 cells. In
conclusion, both human and rat BBB endothelial cells show
little capacity for3H-FA and14C-MTHF apical uptake. Hence,
these cell lines do not appear to be a good model to study the
transport of folates at the BBB.
C 2010 International Union of Biochemistry and Molecular Biology, Inc.
Volume 36, Number 3, May/June 2010, Pages 201–209 ?
Keywords: folate, methyltetrahydrofolate, blood-brain barrier,
The blood–brain barrier (BBB) is constituted by the brain
microvessel endothelial cells, which possess specialized fea-
tures such as tight intercellular junctions that limit paracellu-
lar permeability under physiological conditions, and that
express a unique pattern of receptors, transporters, and
nonselective drug export pumps that protect the CNS from a
very large variety of potentially harmful hydrophobic com-
pounds [1,2]. Hence, the BBB is essential for maintenance of
the homeostasis of the CNS. Although the importance of the
BBB is well-recognized, there is a paucity of information
concerning the mechanisms of transport across this barrier,
especially when compared with other organs such as the
kidney, liver, and intestine. The reason for this lack of knowl-
edge derives mainly from inadequate techniques available
to study transport across the BBB. Most of the transport
studies have been performed using in vivo animal models,
primary cultures of brain capillary endothelial cells, or cocul-
tures of brain microvessel endothelial cells and astrocytes.
However, all these models present serious disadvantages
[1,2]. In this context, the recent establishment of immortal-
ized brain endothelial cell culture systems seems very inter-
esting. The RBE4 and the hCMEC/D3 cell lines are two
immortalized cerebral microvascular endothelial cell lines
that retain most of the morphological characteristics of rat
and human BBB endothelial cells, respectively, [1,2].
Folates (vitamin B9), the generic name given to a family
of water-soluble vitamins, play a critical role in the develop-
ment, function, and repair of the CNS. These one-carbon
donors are required in key biosynthetic processes in mam-
malian cells (including those in the CNS): 1) the de novo
synthesis of purine and thymidylate precursors of nucleic
acids, being thus essential for the de novo production of
RNA and DNA; 2) the initiation of protein synthesis in mito-
chondria, and 3) the biosynthesis of methionine, the precur-
sor of S-adenosylmethionine, which is required for methyla-
tion of DNA, histones, and neurotransmitters [3–6]. The
importance of folates in CNS homeostasis is supported by
several observations. First, the deficiency of folates is impli-
cated in a number or neurological disorders including
Alzheimer’s disease [7,8], Parkinson’s disease , and cere-
brovascular dysfunction [10,11], although some conflicting
clinical evidence has been found for some of these associa-
tions, namely, cerebrovascular disease and dementia-type
disorders [12,13]. Also, cerebral folate deficiency has been
*Address for correspondence: Fa ´tima Martel, Ph.D., Department of Biochemistry,
Faculty of Medicine of Porto, 4200-319 Porto, Portugal. Tel.: þ351 22 5513624; Fax:
þ351 22 5513624; E-mail: email@example.com.
Received 10 November 2009; accepted 1 February 2010
Published online 15 March 2010 in Wiley InterScience
associated with some syndromes. First, hereditary folate
malabsorption, which is associated with defects in both in-
testinal folate absorption and folate transport into the brain
 and which was recently shown to result from mutation in
the proton-coupled folate transporter (PCFT) [14,15]. Second,
decreased folate levels in the cerebrospinal fluid with nor-
mal folate blood levels and which was recently found to be
associated with the presence of autoantibodies against fo-
late receptor a (FRa) present on the choroid plexus [16,17]
or with mutations in FRa .
Because the mechanisms involved in the transport of
folates at the BBB are still not very well-characterized ,
the aim of this study was to characterize the transport of
both folic acid (FA) and methyltetrahydrofolic acid (MTHF) at
the BBB level. For this, we used two distinct cell lines: an
immortalized cell line of rat capillary cerebral endothelial
cells (RBE4 cells) and an immortalized cell line of human
cerebral endothelial cells (hCMEC/D3 cells).
2. Materials and methods
2.1. Cell culture
The RBE4 cell line was kindly supplied by Dr. Franc ¸oise Roux
(INSERM U. 26, Ho ˆpital Fernand Widal, Paris, France) and
was used between passage number 68–83. The cells were
maintained in a humidified atmosphere of 5% CO2to 95%
air and were grown in Minimum Essential Medium/Ham’s
F10 (1:1; Sigma, St. Louis, MO) supplemented with 300 ng
mL?1neomycine, 1 ng mL?1basic fibroblast growth factor,
10% fetal calf serum, 25 mM HEPES, 100 units mL?1penicil-
lin, 100 lg mL?1streptomycin, and 0.25 lg mL?1amphoteri-
cin B (all from Sigma). Culture medium was changed every 2
to 3 days and the culture was split every 7 days. For subcul-
turing, the cells were removed enzymatically (0.25% trypsin-
EDTA, 5 min, 37?C), split 1:5, and subcultured in plastic cul-
ture dishes (21 cm2; ? 60 mm; TPPV
land). For the transport experiments, the RBE4 cells were
seeded in 24-well plastic cell culture clusters (2 cm2; ? 16
the cells formed a monolayer, corresponding to 7–10 days
after the initial seeding. Each square centimeter contained
about 200 lg cell protein.
The hCMEC/D3 cell line was kindly supplied by Drs. P.-
O. Couraud, I. Romero, and B. Weksler (Institut Cochin,
Centre National de la Recherche Scientifique UMR 8104,
INSERM, U567, Universite ´ Rene ´ Descartes, Paris, France)
and was used between passage number 30–37. The cells
were maintained in a humidified atmosphere of 5% CO2to
95% air and were grown in EBM-2 medium (Microvascular
Endothelial Cell Medium-2; Clonetics, Cambrex BioScience,
Wokingham, UK) supplemented with 2.5% heat-inactivated
fetal calf serum, 10 mM HEPES, 1 ng mL?1bFGF, 100 units
mL?1penicillin, 100 lg mL?1streptomycin, and 0.25 lg
mL?1amphotericin B (all from Sigma), VEGF, IGF-1, EGF, and
hydrocortisone, according to the manufacturer’s instructions
(EGM-2 MV bullet Kit). Culture medium was changed every
R, Trasadingen, Switzer-
R). Uptake studies were performed 5–8 days after
2–3 days and the culture was split every 3–4 days. For sub-
culturing, the cells were removed enzymatically (0.25% tryp-
sin-EDTA, 15 sec, 37?C), split 1:4, and subcultured in previ-
ously collagen-coated plastic culture dishes (21 cm2; ? 60
For the transport experiments, the hCMEC/D3 cells
were seeded in previously collagen-coated 24-well plastic
culture clusters (2 cm2; ? 16 mm; TPPV
tured in EBM-2 medium supplemented 2.5% heat-inactivated
fetal calf serum, 1.4 lM hydrocortisone, 1 ng mL?1bFGF, 10
mM HEPES, 100 units mL?1penicillin, 100 lg mL?1strepto-
mycin, and 0.25 lg mL?1amphotericin B (all from Sigma).
Uptake studies were performed 8–9 days after the initial
R) and they were cul-
2.2. Luminal inward transport studies
The transport experiments were performed in buffer with the
following composition (in mM): 125 NaCl, 4.8 KCl, 1.2
KH2PO4, 12.5 HEPES-NaOH, 12.5 MES, 1.2 MgSO4, 1.2 CaCl2,
and 5.6 D(þ)glucose, pH 7.5. Initially, the growth medium
was aspirated and the cells were washed twice with buffer
at 37?C; then the cell monolayers were preincubated for 20
min in buffer at 37?C. Uptake was initiated by the addition
of 0.3 mL medium at 37?C containing3H-FA (25 or 50 nM)
or14C-MTHF (5 lM). Incubation was stopped by placing the
cells on ice and rinsing the cells with 0.5 mL ice-cold buffer.
The cells were then solubilized with 0.3 mL 0.1% (v/v) Triton
X-100 (in 5 mM Tris HCl, pH 7.4), and placed at 37?C over-
night. Radioactivity in the cells was measured by liquid scin-
Effect of drugs. Drugs to be tested were present during
both the preincubation and incubation periods.
Effect of pH of the external medium. To determine the influ-
ence of external pH on the uptake3H-FA or14C-MTHF, cells
were preincubated and incubated with buffer in which the
pH varied from 5 to 8.5.
Effect of ionic composition of the external medium. To
study the influence of external Naþand Cl?on the uptake
bated in NaCl-free buffer, NaCl (corresponding to 125 mM)
being isotonically replaced with either lithium chloride or
choline chloride, or with sodium fluoride, respectively.
14C-MTHF, cells were preincubated and incu-
2.3. Protein determination
The protein content of cell monolayers was determined as
described by Bradford , with human serum albumin as
Total RNA was extracted from hCMEC/D3 and RBE4 cells
using the TripureV
isolation reagent, according to the
manufacturer’s instructions (Roche Diagnostics, Germany).
Before RNA extraction, the culture medium was aspirated
and the cells were suspended in buffer (with the composi-
tion shown above).
Total RNA was extracted with phenol–chloroform, precipi-
tated with ethanol and dissolved in water. For cDNA synthesis,
5 lg of RNA thus prepared was incubated at 25?C for 10 min,
42?C for 50 min, and 70?C for 15 min, in a total volume of
20 lL, with 200 U of Superscript II reverse transcriptase (Invi-
trogen Corporation, Carlsbad, CA) in 10 lM random hexamers
(Sigma), 0.375 mM dNTP (Bioron GmbH, Ludwigshafen,
Germany), 3 mM MgCl2, 100 mM KCl, 50 mM Tris–HCl (pH 8.3;
(RNaseOUT; Invitrogen Corporation). The cDNA thus obtained
was then incubated at 37?C for 20 min with 0.5 mg mL?1
DNase-free RNase H (Invitrogen Corporation) to degrade
unreacted RNA. Using 4 lL of this preparation, PCR was per-
formed. The PCR mixture (50 lL) contained 0.5 lM per primer,
0.2 mM per dNTP, 2.3 mM MgCl2,and 2 U of DyNAzyme II (Finn-
zymes Oy, Keilaranta, Espoo, Finland).
The primer pairs used for amplification and the pre-
dicted size of PCR products were as follows: 50-AGA TAC GGC
CAG GGG AGA GCT TCA T-30(forward) and 50-GTA GGA GGA
ATA GGC GAT GCG CGC-30(reverse) for human reduced folate
transporter (RFC) (hRFC; 299 bp); 50-CTC TAC GAG TGC TCC
CCC AAC TTG-30(forward) and 50-GTC AGC TGA GCA GCC ACA
GCA GCA-30(reverse) for human FRa (hFRa; 460 bp); 50-ATG
CAG CTT TCT GCT TTG GT-30(forward) and 50-GGA GCC ACA
TAG AGC TGG AC-30(reverse) for human PCFT (hPCFT; 100
bp); 50-ACT GGC GTC TTC ACC ACC AT-30(forward) and 50-TCC
ACC ACC CTG TTG CTG TA-30(reverse) for human glyceralde-
hyde 3-phosphate dehydrogenase (hGAPDH; 682 bp); 50-TGG
TAT CGT GGA AGG ACT CA-30(forward) and 50-AGT GGG TGT
CGC TGT TGA AG-30(reverse) for human GAPDH (hGAPDH;
371 bp); 50-GAA CGT CCG GCA ACC ACA G-30(forward) and
50-GAT GGA CTT GGA GGC CCA G-30(reverse) for rat RFC
(rRFC; 489 bp); 50-ATG AGT GTT CCC CGA ACT TG-30(forward)
and 50-GCA TAG AAC CTC GCC ACT TC-30(reverse) for rat FRa
(rFRa; 370 bp); 50-CCG CCA TCA CCG ATC CAT T-30(forward)
and 50-GCT GGT GCA CTG GGC TTT AGG-30(reverse) for rat
PCFT (rPCFT; 712 bp); 50-CAC TGT GCC CAT CTA TGA GGG-30
(forward) and 50-TCC ACA TCT GCT GGA AGG TGG-30(reverse)
for rat b-actin (rb-actin; 588 bp); and 50-CCA TCA CCA TCT
TCC AGG AG-30(forward) and 50-CCT GCT TCA CCA CCT TCT
TG-30(reverse) for rat GAPDH (rGAPDH; 576 bp). The thermo-
cycling conditions for hRFC were 95?C for 5 min (1 cycle),
95?C for 60 sec, 60?C for 90 sec, 72?C for 90 sec (30
cycles), and 72?C for 10 min (1 cycle). For hFRa, thermocy-
cling conditions were 94?C for 5 min (1 cycle), 94?C for 60
sec, 65?C for 90 sec, 72?C for 90 sec (35 cycles), and 72
?C for 10 min (1 cycle). For hPCFT, thermocycling conditions
were 95?C for 5 min (1 cycle); 95?C for 60 sec, 64?C for
45 sec, 72?C for 90 sec (35 cycles); and 72?C for 10 min
(1 cycle). For rRFC and rFRa, the thermocycling conditions
were 95?C for 2 min (1 cycle), 94?C for 60 sec, 68?C for 60
(1 cycle). For rPCFT, thermocycling conditions were 94?C for
2 min (1 cycle); and 94?C for 20 sec, 60?C for 20 sec, 72?C for
?C), 10 mM dithiothreitol, and 40 U of RNase inhibitor
?C for 60 sec (35 cycles), and 72
?C for 10 min
40 sec (35 cycles). Individual PCR reaction products were run
on 1.6% agarose gel (except for hPCFT, which were run on 2.5%
agarose gel), and visualized with an ultraviolet transilluminator
reaction products were recorded in a GelDOC-ItTMImaging Sys-
tem camera with the appropriate filters for UV light.
R, Cambridge, UK) using ethidium bromide staining. PCR
2.5. Calculations and statistics
For the analysis of the time course of
uptake, the parameters of Eq. (1) were fitted to the experi-
mental data by a nonlinear regression analysis, using a com-
puter-assisted method :
AðtÞ ¼ Kin=Koutð1 ? e?kout:tÞ
A(t) represents the accumulation of3H-FA or14C-MTHF at
time t, kinand koutthe rate constants for inward and outward
transport, respectively, and t the incubation time. Amax is
defined as the accumulation at steady-state (t ! 1). kinis
given in pmol mg protein?1min?1and koutin min?1. To obtain
clearance values, kinwas converted to lL mg protein?1min?1.
Arithmetic means are given with SEM. Statistical signif-
icance of the difference between various groups was eval-
uated by one-way analysis of variance (ANOVA test) followed
by the Newman-Keuls test. For comparison between two
groups, Student’s t-test was used. Differences were consid-
ered to be significant when P < 0.05.
3H-FA ([3,5,7,9-3H]-FA sodium salt; specific activity 30 Ci
mmol?1) (Biotrend, Chemikalien GmbH, Koeln, Germany),14C-5-
MTHF (5-[14C]-methyl-tetrahydrofolic acid barium salt, specific
activity 52 mCi mmol?1) (Amersham Pharmacia Biotech, Buck-
inghamshire, UK); 2,4-dinitrophenol, folic acid, DIDS (4,40-diiso-
thiocyanatostilbene-2,20-disulfonic acid disodium salt), SITS
(4-acetamido-40-isothiocyanato-2,20-stilbenedisulfonic acid dis-
sodium salt hydrate), MTHF (5-methyltetrahydrofolic acid
disodium salt), MTX (amethopterin), cytochalasin D (from Zygo-
sporium mansonni), monensin sodium, HEPES (N-2-hydroxy-
ethylpiperazine-N0-2-ethanesulfonic acid), MES (2-[N-morpholi-
no]ethanesulfonic acid) hydrate), sodium azide, trypsin-EDTA
solution (Sigma); DMSO (dimethylsulfoxide), Triton X-100, Tris
Darmstadt, Germany); AlimtaV
pany, Indianapolis, IN).
Some drugs were dissolved in NaOH 0.1 M, NaOH 0.01
M, NaHCO31 mM, or DMSO. The final concentration of these
solvents in the buffer was 1%. Controls for these drugs were
run in the presence of the respective solvents.
R(Premetrexed) (Eli Lilly and Com-
3.1. Time course of3H-FA and14C-MTHF luminal
uptake by RBE4 cells
In a first series of experiments, we determined the time
course of3H-FA and14C-MTHF luminal uptake by RBE4 cells.
Uptake of folates at the blood-brain barrier
For this, cells were incubated with
14C-MTHF (5 lM) for various periods of time. As shown in
Figs. 1A and 2A, RBE4 cells accumulated3H-FA and14C-MTHF
in a time-dependent way, and uptake of both compounds
was found to be linear with time for up to 2 min of incuba-
tion. So, in most of the subsequent experiments, cells were
incubated with3H-FA or14C-MTHF for 2 min to measure ini-
tial rates of uptake. In some experiments, however, cells
were incubated with either3H-FA or14C-MTHF for 6 min to
measure steady-state accumulation of the compounds.
3H-FA (25 nM) or
3.2. pH-dependence of3H-FA and14C-MTHF
luminal uptake and steady-state accumulation
by RBE4 cells
The pH-dependence of
and steady-state accumulation by RBE4 cells were analyzed
by preincubating and incubating cells in buffer with different
In relation to3H-FA, both uptake of this compound and
its steady-state accumulation showed pH-dependence; inter-
14C-MTHF luminal uptake
estingly, both parameters were significantly increased when
the external pH was decreased to 5.0 or increased to 8.5
(Figs. 1B and 1C).
In relation to14C-MTHF, a distinct effect of pH variation
on uptake and steady-state accumulation of this compound
was observed. Although uptake of
pH-dependence (Fig. 2B), the steady-state accumulation
increased when the external pH increased to 8 and 8.5
14C-MTHF showed no
3.3. Ionic dependence of3H-FA and14C-MTHF
luminal uptake by RBE4 cells
We next analyzed the dependence of
luminal uptake by RBE4 cells in relation to external Naþand
Cl?. Uptake of3H-FA was reduced (by 20–25%) when exter-
nal Naþwas substituted by either Liþor cholineþ. On the
other hand, substitution of Cl?by F?caused an 80%
the other hand, showed neither Naþnor Cl?-dependence
3H-FA uptake (Fig. 1D). Uptake of
Fig. 1. Characteristics of3H-FA luminal uptake by RBE4 cells. A: Time-course of3H-FA uptake by RBE4 cells incubated
at 37?C with 25 nM3H-FA (n 5 6). B,C: pH-dependence of3H-FA uptake by RBE4 cells incubated at 37?C with 25 nM
3H-FA for 2 or 6 min. The extracellular pH in the preincubation and incubation media ranged from 5.5 to 8.0 (n 5
6–12). *Significantly different from uptake at pH 7.5. D: Ionic-dependence of3H-FA uptake by RBE4 cells incubated at
37?C with 50 nM3H-FA for 2 min. NaCl in the preincubation and incubation media was isotonically replaced by either
LiCl, choline chloride (ChCl), or NaF (n 5 5–9). *Significantly different from control (NaCl). Shown are arithmetic
means 6 SEM.
3.4. Energy dependence of3H-FA and14C-MTHF
luminal uptake by RBE4 cells
As shown in Figs. 3A and 4A, uptake of3H-FA and14C-MTHF
showed no energy dependence, as it was not inhibited in
the presence of the metabolic inhibitors sodium azide and
3.5. Effect of drugs on3H-FA and14C-MTHF luminal
uptake by RBE4 cells
In this series of experiments, the effect of a series of com-
pounds, known to affect3H-FA and14C-MTHF uptake in other
cell types, was tested. In relation to
significantly decreased (by 32%) by premetrexed (20 lM)
and significantly increased (by 27%) by cytochalasin D (Fig.
3B). In relation to
decreased in the presence of premetrexed (20 and 50 lM)
(by 30%) and methotrexate 20 lM (by 25%); on the other
hand, cytochalasin D increased it (by 40%) (Fig. 4B).
3H-FA uptake, it was
14C-MTHF uptake, it was significantly
3.6. Time course of3H-FA and14C-MTHF luminal
uptake by hCMEC/D3 cells
In this series of experiments, the time course of3H-FA and
14C-MTHF luminal uptake by hCMEC/D3 cells was deter-
mined. For this, cells were incubated with3H-FA (25 nM) or
14C-MTHF (5 lM) for various periods of time. As shown in
Fig. 5, uptake of
showed only a slight time dependency and a great variability
in the experimental results was observed. Moreover, uptake
of both compounds was not linear with time, even in the
first minute of incubation. So, further experiments with this
cell line were not performed.
14C-MTHF by hCMEC/D3 cells
The expression of RFC, FRa, and PCFT in both RBE4 cells
and hCMEC/D3 cells was analyzed by RT-PCR. Both cell lines
were positive for RFC and negative for FRa. Moreover, PCFT
Fig. 2. Characteristics of14C-MTHF luminal uptake by RBE4 cells. A: Time-course of14C-MTHF uptake by RBE4 cells
incubated at 37?C with 5 lM14C-MTHF (n 5 6). B,C: pH-dependence of14C-MTHF uptake by RBE4 cells incubated at
37?C with 5 lM14C-MTHF for 2 or 6 min. The extracellular pH in the pre-incubation and incubation media ranged from
5.5 to 8.0 (n 5 12). *Significantly different from uptake at pH 7.5. D: Ionic-dependence of14C-MTHF uptake by RBE4
cells incubated at 37?C with 5 lM14C-MTHF for 2 min. NaCl in the in the preincubation and incubation media was
isotonically replaced by either LiCl, choline chloride (ChCl), or NaF (n 5 6–12). *Significantly different from control
(NaCl). Shown are arithmetic means 6 SEM.
Uptake of folates at the blood-brain barrier
mRNA expression was demonstrated in hCMEC/D3 cells but
not in RBE4 cells (Fig. 6).
There are two potential routes of folates entry into the brain:
the BBB at the cerebral vascular endothelium and the blood-
CSF barrier at the choroid plexus. Because the mechanisms
involved in the transport of folates at the BBB are still not
very well-characterized , we decided to determine the
characteristics of3H-FA and14C-MTHF luminal uptake by the
RBE4 and hCMEC/D3 cell lines.
The RBE4 cell line was obtained by transfection of rat
brain microvessel endothelial cells with a plasmid containing
the E1A adenovirus gene . These cells display a non-
transformed endothelial phenotype and express a series of
BBB specific markers (e.g., the enzymes c-glutamyl-trans-
peptidase and alkaline phosphatase, and the transporters
GLUT1, LAT1, P-glycoprotein, and the transferrin receptor
(review by ). The immortalized RBE4 cell line thus
Fig. 3. Characteristics of3H-FA luminal uptake by RBE4
cells incubated at 37?C with 50 nM3H-FA for 2 min. A:
Energy-dependence of3H-FA uptake by RBE4 cells.
Cells were incubated in the absence or presence of
sodium azide (10 mM) or dinitrophenol (10 mM) (n 5
6). B: Effect of drugs on3H-FA uptake by RBE4 cells.
Cells were incubated in the absence or presence of folic
acid 10 lM (FA 10) or 100 lM (FA 100) (n 5 6–9), 5-
methyltetrahydrofolic acid 10 lM (MTHF 10) or 20 lM
(MTHF 20) (n 5 3–15), methotrexate 10 lM (MTX 10) or
20 lM (MTX 20) (n 5 9), premetrexed 20 lM (PTX 20)
or 50 lM (PTX 50) (n 5 6), DIDS 500 lM (DIDS; n 5
9), SITS 500 lM (SITS; n 5 9), cytochalasin D 1 lg/mL
(Cyt D; n 5 9) or monensin 10 lM (Mon; n 5 9).
*Significantly different from control. Shown are
arithmetic means 6 SEM.
Fig. 4. Characteristics of14C-MTHF luminal uptake by
RBE4 cells incubated at 37?C with 5 lM14C-MTHF for 2
min. A: Energy-dependence of14C-MTHF uptake by
RBE4 cells. Cells were incubated in the absence or
presence of sodium azide (10 mM) or dinitrophenol (10
mM) (n 5 6). B: Effect of drugs on14C-MTHF uptake by
RBE4 cells. Cells were incubated in the absence or
presence of folic acid 500 lM (FA 500) (n 5 6),
5-methyltetrahydrofolic acid 500 lM (MTHF 500) or
1,000 lM (MTHF 1000) (n 5 9), methotrexate 10 lM
(MTX 10) or 20 lM (MTX 20) (n 5 6–8), premetrexed
20 lM (PTX 20) or 50 lM (PTX 50) (n 5 6–8), DIDS 500
lM (DIDS; n 5 9), SITS 500 lM (SITS; n 5 9),
cytochalasin D 1 lg/mL (Cyt D; n 5 12), or monensin 10
lM (Mon; n 5 9).*Significantly different from control.
Shown are arithmetic means 6 SEM.
preserves many features of the in vivo brain endothelium
and hence is of interest as a potential in vitro model of the
As to the hCMEC/D3 cell line, it was recently estab-
lished and represents the first stable, fully characterized,
well-differentiated immortalized human cerebral microvascu-
lar endothelial cell line . hCMEC/D3 cells retain most of
the morphological characteristics of human BBB endothelial
cells, express normal endothelial markers, and demonstrate
BBB characteristics, including tight junctional proteins and
the capacity to actively exclude drugs . This cell line thus
represents a promising, reliable in vitro model of the human
RT-PCR analysis demonstrates mRNA expression of
RFC, but not of FRa or PCFT, in RBE4 cells. In contrast,
mRNA expression of both RFC and PCFT, but not of FRa, was
demonstrated in hCMEC/D3 cells. To our knowledge, this is
the first report of RFC, PCFT, and FRa mRNA expression anal-
ysis in human and rat brain endothelial cells. Interestingly
enough, our results confirm previous findings by others,
namely, the expression of RFC in mice BBB , and the
lack of expression of FRa in both human and mice BBB
Our characterization results can be summarized as fol-
lows. Uptake of3H-FA by RBE4 cells was 1) stimulated both
at an acidic (5.0) and at an alkaline (8.5) pH (the same was
dependent and stimulated when F?substituted for Cl?, 3)
energy independent, 4) inhibited by premetrexed and stimu-
lated by cytochalasin D, 5) not affected by MTHF, DIDS,
SITS, methotrexate, monensin, and FA. On the other hand,
uptake of14C-MTHF was 1) pH independent, but its steady-
increased (to 8–8.5); 2) Naþindependent and Cl?independ-
ent, 3) energy independent, 4) inhibited by premetrexed and
methotrexate and stimulated by cytochalasin D, 5) not
affected by FA, DIDS, SITS, monensin, and MTHF.
Because RFC (but neither PCFT nor FRa) is expressed
in RBE4 cells, a comparison between the characteristics of
3H-FA and14C-MTHF luminal uptake and RFC-mediated trans-
port was made. Some of the characteristics of both
and14C-MTHF luminal uptake are compatible with RFC-medi-
ated transport (namely, in relation to3H-FA uptake, its inhi-
bition by premetrexed and its stimulation by F?, and in rela-
independence and its inhibition by methotrexate and preme-
trexed) . However, many other characteristics of both
3H-FA and14C-MTHF luminal uptake are not compatible with
the involvement of this transporter (namely, the lack of in-
hibitory effect of DIDS, SITS, MTHF, and FA on both
uptake). Hence, based in these results, we cannot conclude
on the transporter(s) involved in uptake of these two com-
pounds by RBE4 cells. However, based on the expression
results for folates transporters, we predict that uptake of
folates at the BBB will most probably involve RFC. This
3H-FA steady-state accumulation); 2) Naþ
14C-MTHF uptake, its pH independence and Naþ
14C-MTHF uptake, and the pH-dependency of
Fig. 5. Characteristics of3H-FA and14C-MTHF luminal
uptake by hCMEC/D3 cells. A: Time course of3H-FA
uptake by hCMEC/D3 cells incubated at 37?C with 25
nM3H-FA (n 5 7). B: Time course of14C-MTHF uptake
by RBE4 cells incubated at 37?C with 5 lM14C-MTHF
(n 5 6).
Fig. 6. RT-PCR analysis of RFC, FRa, and PCFT mRNA
expression in (A) RBE4 cells and (B) hCMEC/D3 cells.
The housekeeping genes, GAPDH or b-actin, were
amplified as the control.
Uptake of folates at the blood-brain barrier
conclusion is based on the observation that RFC, a trans-
porter with a neutral pH optimum, is expressed by both
RBE4 and hCMEC/D3 cells. Moreover, although expressed by
hCMEC/D3 cells, PCFT functions optimally at an acidic pH,
and there is no evidence that the pH at the BBB interface is
A last point deserves some reflection. From our results,
we conclude that both RBE4 cells and hCMEC/D3 cells have
little capacity for
sion is based in two main reasons: 1) the amount of radioac-
tive compounds taken up by these cells is very small, when
compared with other cell types we have previously used
(e.g., Caco-2 cells, BeWo cells) and 2) both
MTHF uptake were not affected by some well-known sub-
strates/inhibitors of transporters of folates (DIDS, SITS,
MTHF, FA, methotrexate), raising the hypothesis that a frac-
tion of3H-FA and14C-MTHF measured as ‘‘uptake’’ by both
cell lines corresponds to unspecific adsorption of the radio-
active compounds to the cell membrane.
In conclusion, both human (hCMEC/D3) and rat (RBE4)
BBB endothelial cells show little capacity for3H-FA and14C-
MTHF apical uptake. This low capacity for both
14C-MTHF uptake by RBE4 and hCMEC/D3 cells is not in
agreement with the high delivery of MTHF to brain paren-
chyma, thought to occur predominantly through MTHF
extraction from blood at the BBB level . Hence, we con-
clude that these cell lines are not a good model to study
the transport of folates at the BBB.
14C-MTHF uptake. This conclu-
This work was supported by Fundac ¸a ˜o para a Cie ˆncia e a
Tecnologia (FCT) and Programa Cie ˆncia, Tecnologia e Inova-
c ¸a ˜o do Quadro Comunita ´rio de Apoio (PTDC/SAU-FCF/
67805/2006). The authors would like to thank Dr. Franc ¸oise
Roux (INSERM U. 26, Ho ˆpital Fernand Widal, Paris, France)
for the generous supply of RBE4 cells and Drs. P.-O. Cou-
raud, I. Romero, and B. Weksler (Institut Cochin, Centre
National de la Recherche Scientifique UMR 8104, INSERM,
U567, Universite ´ Rene ´ Descartes, Paris, France) for the gen-
erous supply of hCMEC/D3 cells.
 Roux, F. and Couraud, P. O. (2005) Rat brain endothelial cell lines for
the study of blood.brain barrier permeability and transport functions.
Cell Mol. Neurobiol. 25, 41–58.
 Weksler, B. B., Subileau, E. A., Perrie `re, N., Charneau, P., Holloway, K.,
Leveque, M., Tricoire-Leignel, H., Nicotra, A., Bourdoulous, S., Turowski,
P., Male, D. K., Roux, F., Greenwood, J., Romero, I. A., and Couraud, P.
O. (2005) Blood-brain barrier-specific properties of a human adult brain
endothelial cell line. FASEB J. 19, 1872–1874.
 Herbert, V. (1999) Folic acid. In Modern Nutrition in Health and Disease,
ed. 9. (Shils, M. E., Olson, J. A., Shike, M., Ross, A. H., eds.). pp.433–
446, Lippincott Williams & Wilkins, London.
 Lucock, M. (2000) Folic acid: nutritional biochemistry, molecular biol-
ogy, and role in disease processes. Mol. Gen. Metab. 71, 121–138.
 Worthington-Roberts, B. S. (1999) Nutrition. In Cherry and Merkatz’s
Complications of Pregnancy, ed. 5. (Cohen, W. R., Cherry, S. H., Mer-
katz, I. R., eds.). pp.17–49, Lippincott Williams & Wilkins, London.
 Zhao, R., Matherly, L. H., and Goldman, I. D. (2009) Membrane trans-
porters and folate homeostasis: intestinal absorption and transport
into systemic compartments and tissues. Exp. Rev. Mol. Med. 11, e4.
 Serot, J. M., Christmann, D., Dubost, T., Bene, M. C., and Faure, G. C.
(2001) CSF-folate levels are decreased in late-onset AD patients. J. Neu-
ral. Transm. 108, 93–99.
 Snowdon, D. A., Tully, C. L., Smith, C. D., Riley, K. P., and Markesbery,
W. R. (2000) Serum folate and the severity of atrophy of the neocortex
in Alzheimer disease: findings from the Nun study. Am. J. Clin. Nutr. 71,
 Duan, W., Ladenheim, B., Cutler, R. G., Kruman, I. I., Cadet, J. L., and
Mattson, M. P. (2002) Dietary folate deficiency and elevated homocys-
teine levels endanger dopaminergic neurons in models of Parkinson’s
disease. J. Neurochem. 80, 101–110.
 Endres, M., Ahmadi, M., Kruman, I., Biniszkiewicz, D., Meisel, A., and
Gertz, K. (2005) Folate deficiency increases postischemic brain injury.
Stroke 36, 321–325.
 Giles, W. H., Kittner, S. J., Anda, R. F., Croft, J. B., and Casper, M. L.
(1995) Serum folate and risk for ischemic stroke. First National Health
and Nutrition Examination Survey epidemiologic follow-up study. Stroke
 Aisen, P. S., Schneider, L. S., Sano, M., Diaz-Arrastia, R., van Dyck, C.
H., Weiner, M. F., Bottiglieri, T., Jin, S., Stokes, K. T., Thomas, R. G.,
Thal, L. J., Alzheimer Disease Cooperative Study. (2008) High-dose B
vitamin supplementation and cognitive decline in Alzheimer disease: a
randomized controlled trial. JAMA 300, 1774–1783.
 Maron, B. A. and Loscalzo, J. (2009) The treatment of hyperhomocystei-
nemia. Annu. Rev. Med. 60, 39–54.
 Qiu, A., Jansen, M., Sakaris, A., Min, S., Chattopadhyay, S., Tsai, E.,
Sandoval, C., Zhao, R., Akabas, M., and Goldman, I. D. (2006) Identifica-
tion of an intestinal folate transporter and the molecular basis for he-
reditary folate malabsorption. Cell 127, 917–928.
 Zhao, R., Min, S. H., Qiu, A., Sakaris, A., Goldberg, G. L., Sandoval, C.,
Malatack, J. J., Rosenblatt, D. S., Goldman, I. D. (2007) The spectrum of
mutations in the PCFT gene, coding for an intestinal folate transporter,
that are the basis for hereditary folate malabsorption. Blood 110,
 Ramaekers, V. T. and Blau, N. (2004) Cerebral folate deficiency. Dev.
Med. Child Neurol. 46, 843–851.
 Ramaekers, V. T., Rothenberg, S. P., Sequeira, A. J., Opladen, T., Blau,
N., Quadros, E. V., and Slhub, J. (2005) Autoantibodies to folate recep-
tors in the cerebral. N. Engl. J. Med. 52, 1985–1991.
 Steinfeld, R., Grapp, M., Kraetzner, R., Dreha-Kulaczewski, S., Helms,
G., Dechent, P., Wevers, R., Grosso, S., and Ga ¨rtner, J. Folate receptor
alpha defect causes cerebral folate transport deficiency: a treatable
neurodegenerative disorder associated with disturbed myelin metabo-
lism. Am. J. Hum. Genet. 85, 354–363.
 Wu, D. and Pardridge, W. M. (1999) Blood-brain barrier transport of
reduced folic acid. Pharm. Res. 16, 415–419.
 Bradford, M. M. (1976) A rapid method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem. 72, 248–254.
 Muzyka, A., Tarkany, O., Yelizanof, V., Sergienko, U., and Boichuk, A.
(2005) Non-Linear Regression (Curve Fit), GraphPad Prism for Windows,
San Diego, CA.
 Roux, F., Durieu-Trautmann, O., Chaverot, N., Claire, M., Mailly, P.,
Bourre, J. M., Strosberg, A. D., and Couraud, P. O. (1994) Regulation of
gamma-glutamyl transpeptidase and alkaline phosphatase activities in
immortalized rat brain microvessel endothelial cells. J. Cell Physiol.
 Afonso, P. V., Ozden, S., Prevost, M. C., Schmitt, C., Seilhean, D., Weks-
ler, B., Couraud, P. O., Gessain, A., Romero, I. A., and Ceccaldi, P. E.
(2007) Human blood–brain barrier disruption by retroviral-infected lym-
phocytes: role of myosin light chain kinase in endothelial tight-junction
disorganization. J. Immunol. 179, 2576–2583.
 Andras, I. E., Rha, G., Huang, W., Eum, S., Couraud, P. O., Romero, I.
A., Hennig, B., and Toborek, M. (2008) Simvastatin protects against
amyloid beta and HIV-1 Tat-induced promoter activities of inflammatory
genes in brain endothelial cells. Mol. Pharmacol. 73, 1424–1433.
 Cucullo, L., Couraud, P. O., Weksler, B., Romero, I. A., Hossain, M.,
Rapp, E., and Janigro, D. (2008) Immortalized human brain endothelial
cells and flow-based vascular modeling: a marriage of convenience for
rational neurovascular studies. J. Cereb. Blood Flow Metab. 28,
 Forster, C., Burek, M., Romero, I. A., Weksler, B., Couraud, P. O., and
Drenckhahn, D. (2008) Differential effects of hydrocortisone and TNFal-
pha on tight junction proteins in an in vitro model of the human
blood–brain barrier. J. Physiol. 586, 1937–1949.
 Wang, Y., Raine, A., Narr, K. L., Lencz, T., LaCasse, L., Colletti, P., and Toga, A.
W. (2001) Localization of the murine reduced folate carrier as assessed by
immunohistochemical analysis. Biochim. Biophys. Acta 1513, 49–54.
 Kennedy, M. D., Jallad, K. N., Lu, J., Low, P. S., and Ben-Amotz, D.
(2003) Evaluation of folate conjugate uptake and transport by the cho-
roid plexus of mice. Pharm. Res. 20, 714–719.
 Weitman, S. D., Frazier, K. M., and Kamen, B. A. (1994) The folate re-
ceptor in central nervous system malignancies of childhood. J. Neur.
Oncol. 21, 107–112.
Uptake of folates at the blood-brain barrier