(Received for publication, December 3, 1996, and in revised form, February 18, 1997)
From the Molecular Pharmacology and Therapeutics Program, Mediated folate compound transport inward in
isolated luminal epithelial cells from mouse small intestine was
delineated as pH-dependent and non-pH-dependent
components on the basis of their differential sensitivity to the
stilbene inhibitor, 4,4 Transport of folate compounds into mammalian cells can occur via
carrier-mediated (1-5) as well as receptor-mediated (3-7) mechanisms
depending on the relative level at which each is expressed in the
plasma membrane of these cells. Carrier-mediated mechanisms are
involved (1-4) in the internalization of both natural folates and
their cytotoxic analogues in tumor and normal proliferative tissues.
Thus, their level of expression in these tissues has pharmacological
significance. Carrier-mediated transport of folate compounds among
tumor cells from various tissues in different mammalian species appears
to share (1-4) similar properties. They exhibit (1-4) similar
diversity in saturability for various folate compounds and their
analogues with extremely low saturability for folic acid compared with
folate coenzymes. The properties of folate compound transport in normal
tissues appear to be much more diverse (reviewed in Ref. 2) and often
differ substantially from those characteristic of mechanisms found in
tumor cells. Carrier-mediated transport of folates associated (8-11)
with absorption in the small intestine of rodents appears to be
uniquely different from that in tumor cells (1-4, 12), particularly in
terms of its pH dependence and saturability. For instance, in
absorptive luminal epithelium of the rat small intestine, a mechanism
in the brush-border membrane has been described (11, 13) which exhibits
an optimum at pH 5.5-6 and relatively high saturability (Km < 5 µM) for internalization of
all folate compounds and their analogues examined, including folic
acid. Another carrier-mediated mechanism transporting folate compounds
has been described (14) in the luminal epithelium of mouse small
intestine. This moderately saturable mechanism mediates high capacity
influx and does not appear (14) to be associated with folate
absorption. Since this mechanism is operable (14) at the same level in
both absorptive and nonabsorptive proliferative tissue in the luminal
crypt, it would appear to be localized in the plasma membrane at the
basolateral surface.
Recently, a murine cDNA clone (RFC-1) was isolated (15) by
expression cloning from an L1210 cell cDNA library. This cDNA appears to encode a protein with many of the properties of the tumor
cell, 1-carbon reduced folate transporter. However, expression of the
RFC-1 gene has also been shown (16) to occur in the small intestine of rodents and appears to be under developmental regulation at least in rat intestine. This was based on the finding in the rat of
higher levels of RFC-1 mRNA in mature absorptive cells compared
with that found in immature proliferative cells in the crypt. However,
direct evidence showing that this gene does, in fact, regulate folate
absorption in this tissue was not provided.
The present studies are an extension of these recently reported (16)
and of our own earlier (14) studies of folate compound transport in
luminal epithelial cells isolated from mouse small intestine. In these
new studies, we were able to delineate by kinetic and other means acid
pH-dependent influx of
l,L-5-CH3-H4folate1
from non-pH-dependent influx mediated (14) by the mechanism that appears to operate in the plasma (basolateral) membrane of these
cells. We also provide evidence showing that acid
pH-dependent folate compound transport in brush-border
membrane in mature absorptive cells is inhibited by a folate-based
affinity label and by antibodies against RFC-1-specific peptides.
Folate transport in absorptive epithelial cells from the mouse also
appears to be dependent on the expression of the RFC-1 gene
(15) in the form of a 2.5-kilobase transcript and a 58-kDa transporter
encoded by this transcript and detected by the same folate-based
affinity label and by immunoblotting in the brush-border membrane of
these cells.
The
isolation of luminal epithelial cells from the small intestine of
C57BL/6 × DBA/2 F1 (BD2F1) mice was
carried out at 0-4 °C by a previously published (14) method. These
cells were obtained in the form of four individual fractions varying in
their stage of maturation using marker enzyme analysis (14) to monitor
fractionation. The isolation of purified brush-border membrane from
Fraction I luminal epithelial cells was the same as that described (13, 17) for rat luminal epithelial cells.
Freshly
isolated cells in Fractions I and IV were utilized in experiments
measuring the mediated internalization at 37 °C of
l,L-5-CH3-H4folate using
published procedures (14, 18). The transport buffer (Hepes-sucrose-MgO)
used during these experiments contained 20 mM Hepes and 227 mM sucrose adjusted to the appropriate pH with MgO. A few
experiments were carried out using a physiological salts solution plus
7 mM D-glucose (14) as the transport buffer. Cells in the Hepes-sucrose-MgO buffer were affinity labeled with 50 nM N-hydroxysuccinimide ester of
[3H]aminopterin as described by Henderson and Zevely
(19). The alkaline-washed purified membranes (20) were fractionated
(20) by SDS-polyacrylamide gel electrophoresis and gel slicing, and the
radioactivity in each fraction was determined by scintillation counting.
Antibodies against two murine RFC-1-specific peptides
were prepared in rabbits according to standard protocols (21). Before injection, the peptides were either linked to keyhole limpet hemocyanin (22) or converted to macromolecular size with multiple antigenic peptide technology (23). The peptides prepared consist of 19 amino
acids encoded at the NH2-terminal (Met1 through
Asp19) or COOH-terminal (Ser495 through
Ala512) regions of the murine RFC-1 cDNA. Before use,
the antibodies in the immune serum were purified by immunoaffinity
fractionation with the NH2- and COOH-terminal peptides
linked to cyanogen bromide-activated Sepharose (24). Western blotting
with enhanced chemiluminescence (Amersham) detection was carried out by
a standard procedure (25) following SDS-polyacrylamide gel
electrophoresis of purified plasma membrane (20).
Standard procedures
(26) were used for detection by blotting of RFC-1 mRNA in luminal
intestinal epithelial cell fractions using a murine RFC-1 cDNA
probe (15), a generous gift of Dr. K. H. Cowan. Total RNA was obtained
from these cells by means of RNA STAT-60 (TEL-TEST, Inc.).
Poly(A)+ RNA was subsequently obtained by fractionation on
an oligo(dT) column (27). All blots were normalized with a Total RNA was prepared by two treatments with RNA
STAT-60 according to the manufacturer's (TEL-TEST, Inc.) instructions.
A 5-µg aliquot of methylmercuric hydroxide-treated RNA was used to
synthesize cDNA by a standard procedure (29) and an oligo(dT) primer (Life Technologies, Inc.). Quantitative PCR was carried out with
Amplitag DNA polymerase (Perkin-Elmer) in the recommended buffer, 400 pmol of RFC-1 sense (5-GAGCCTTTAGGTTAGG-3 l,L-[3H]-5-CH3-H4folate
(specific activity, 30 Ci/mmol) and [3H]aminopterin
(specific activity, 10-12 Ci/mmol) were purchased from Moravek
Biochemicals (City of Industry, CA)
l,L-5-CH3-H4folate was
purchased from Sigma Biochemical (St. Louis, MO). All other reagents
were reagent grade. Radiolabeled folate compounds were analyzed for
purity (>95%) by HPLC (30) before use or repurified by HPLC (30).
The velocity of initial influx
of
l,L-[3H]-5-CH3-H4folate
was determined with Fraction I cells suspended in transport buffer with
150 µM DIDS. At this concentration of the stilbene,
influx by the moderately saturable system previously described (14) for
these cells was almost completely inhibited. Also, the concentration of
the folate utilized (2.5 µM) was >50-fold lower than the
apparent Ki determined (14) for inhibition by this
folate of folate compound influx by this moderately saturable system.
Data shown in Fig. 1A document rapid
internalization of
l,L-[3H]-5-CH3-H4folate
under these conditions in a pH-dependent manner. A
steady-state level of internalization was achieved within 5-10 min at
37 °C at pH 6.2 or 7.4. However, initial influx and steady-state level achieved were both 4-fold greater at pH 6.2. Under the same conditions at pH 6.2, the initial influx and steady-state level of this
folate achieved in Fraction IV cells (Fig. 1B) were
severalfold lower. Influx mediated by the highly saturable,
pH-dependent system in Fraction IV cells is probably even
lower than would appear from these data in view of the residual influx
mediated by the moderately saturable system in contaminating Fraction I
cells that would be expected to be present in the Fraction IV
preparation.
Data on the concentration response for initial influx at 37 °C of
l,L-[3H]-5-CH3-H4folate
in Fraction I and IV cells is shown in Fig. 2A. At pH 6.2, initial influx exhibits
complex saturation kinetics in the form of highly saturable and
moderately saturable components. The highly saturable component was
decreased at pH 7.4 in Fraction I cells and in Fraction IV cells at pH
6.2 or pH 7.4 (data not shown). Moreover, in the presence of 150 µM DIDS, only a highly saturable component of influx is
observed at pH 6.2 in Fraction I cells. A double-reciprocal plot of
some of the data obtained in this type of experiment carried out over a
wider concentration range in the presence an absence of 150 µM DIDS is shown in Fig. 2B. It can be seen
that the two saturable components of influx observed in the absence of
DIDS (Fig. 2B) are converted to a single highly saturable
component in the presence of 150 µM DIDS. From the data
presented in Fig. 2B, an apparent Km and
Vmax for each saturable component at pH 6.2 or
at pH 7.4 (not shown) in Fraction I cells could be obtained. In
Fraction I cells, DIDS-insensitive influx of
l,L-[3H]-5-CH3-H4folate
exhibits (Table I) an apparent Km in
the low micromolar range and an extremely low
Vmax compared with DIDS-sensitive influx of this
folate compound. Only influx Vmax exhibited pH
dependence, with the higher value for Vmax
occurring in the acidic range. In contrast, DIDS-sensitive influx of
this folate compound exhibits both a high Km and a
high Vmax, neither of which are pH dependent.
Therefore, at the low micromolar range of concentration or below,
influx of
l,L-[3H]-5-CH3-H4folate
by the DIDS-insensitive system would predominate in Fraction I cells,
but only at pH 6.2. While at pH 7.4 and at the high micromolar range,
DIDS-sensitive influx would predominate. The data in Table I also
document a much lower Vmax for
l,L-[3H]-5-CH3-H4folate
influx at pH 6.2 in Fraction IV cells in the presence of DIDS.
Kinetic properties of mediated
The values shown are averages ± S.E. for influx by Fraction I or
Fraction IV cells in 3-5 separate experiments. Influx by the high and
low affinity systems was delineated by measurements made in the
presence and absence of 150 µM DIDS. Other experimental details are provided in the text.
Graduate School of
Medical Sciences,
-diisothiocyanatostilbene-2,2
-disulfonic acid.
pH dependence was manifested as higher maximum capacity (Vmax) for influx of
l,L-5-CH3-H4folate at
acidic pH compared with neutral or alkaline pH with no effect on
saturability (Km). The pH-dependent
component was relatively insensitive to inhibition by
4,4
-diisothiocyanatostilbene-2,2
-disulfonic acid and highly saturable
(Km or Ki = 2 to 4 µM) in the case of folic acid, folate coenzymes, and
4-aminofolate analogues as permeants or inhibitors. The
non-pH-dependent component was highly sensitive to
4,4
-diisothiocyanatostilbene-2,2
-disulfonic acid and poorly and
variably saturable (Km or Ki = 20 to >2000 µM) with respect to these folate compounds.
Only the pH-dependent transport component was
developmentally regulated, showing much higher maximum capacity for
l,L-5-CH3-H4folate
influx in mature absorptive rather than proliferative crypt cells. The increase in pH-dependent influx during maturation was
associated with an increase in RFC-1 gene expression in the
form of a 2.5-kilobase RNA transcript and 58-kDa brush-border membrane
protein detected by folate-based affinity labeling and with anti-mouse
RFC-1 peptide antibodies. The size of this protein was the same as that
encoded by RFC-1 mRNA. The treatment of mature absorptive cells
with either the affinity label or the anti-RFC-1 peptide antibodies
inhibited influx of
l,L-[3H]-5-CH3-H4folate
in a concentration-dependent manner. These results strongly
suggest that pH-dependent folate absorption in this tissue is regulated by RFC-1 gene expression.
Isolation of Luminal Epithelial Cell Fractions
-actin
probe (28). Labeling of each probe was by random priming (Boehringer Mannheim) using [
-32P]dCTP (3000 Ci/mmol) and 10 ng of
insert.
) and antisense (5
-CAAGCACCTCCGATAG-3
) primers, 200 µM dNTPs, and 10 µCi [32P]dCTP in a total volume of 50 µl. In a
parallel reaction, sense (5
-TATCAGCACTGGATCG-3
) and antisense
(5
-TTACAGGTGTCGATGC-3
)
-actin primers were used. After the initial
denaturation step of 5 min at 95 °C, 30 cycles of 1.5 min at
95 °C, 1 min at 60 °C, and 1.5 min at 72 °C, respectively,
were carried out. After this, the reaction was extended for 10 min at
72 °C. The relative amount of product formation was determined by
transblotting (26) to Nytran-Plus membrane (Schleicher and Schuell) and
radioautography. The amount of RFC-1 and
-actin cDNA included in
the reaction sustained amplification in the linear range of product
formation at the time the amount of each product was compared.
Kinetic Delineation between Mechanisms Mediating
l,L-[3H]-5-CH3-H4folate
Influx in Luminal Epithelial Cells
Fig. 1.
Time course for mediated accumulation at
37 °C of
l,L-[3H]-5-CH3-H4folate
by luminal epithelial cells from mouse small intestine. A,
influx by Fraction I cells at pH 6.2 and pH 7.4. B, influx by Fraction I and Fraction IV cells at pH 6.2; ext,
external. Data are averages from three experiments with S.E. <±14%.
Other experimental details are provided in the text.
[View Larger Version of this Image (23K GIF file)]
Fig. 2.
Concentration response for mediated
accumulation at 37 °C of
l,L-[3H]-5-CH3-H4folate
by Fraction I and Fraction IV cells. A, influx following 1 min of incubation at different concentrations of
l,L-[3H]-5-CH3-H4folate
in Fraction I cells at pH 6.2 ± 150 µM DIDS or pH
7.4 and in Fraction IV cells at pH 6.2. B, double-reciprocal plot of data on influx at pH 6.2 ± 150 µM DIDS;
ext, external. Other experimental details are provided in
the text. Data are averages from three experiments with S.E.
<±16%.
[View Larger Version of this Image (28K GIF file)]
,L-[3H]-5-CH3-H4 folate influx
in luminal epithelial cells of mouse small intestine
Fraction
DIDS (150 µM)
pH
Km
Vmax
µM
pmol/min/mg
protein
I
+
6.2
1.8
± 0.4
0.8 ± 0.2
7.4
2.3 ± 0.2
0.2
± 0.05
6.2
128 ± 23
18 ± 5
7.4
115 ± 20
16 ± 4
IV
+
6.2
1.9 ± 0.3
0.13 ± 0.03
6.2
119 ± 18
19 ± 4
The two systems delineated above on the basis of inhibition by DIDS and pH dependence were also distinguishable by their interaction with various folate compounds as inhibitors of l,L-[3H]-5-CH3-H4folate influx. In the presence of 150 µM DIDS, influx of this folate by the DIDS-insensitive system was inhibited (Table II) by 5-methyl- or 5-formyl-substituted H4folate as well as folic acid in the low micromolar range, yielding values for Ki of 2-4 µM. Similar values for Ki were obtained with the 4-aminofolate analogues, aminopterin and methotrexate. These values are consistent with other values for Ki obtained when most of these same compounds were used as inhibitors of folic acid influx by brush-border membrane vesicles in studies by others (13). In contrast, inhibition by these same compounds of l,L-[3H]-5-CH3-H4folate influx by the DIDS-sensitive system (see Table II) was more variable and much less in extent.
|
Fraction I cells were affinity
labeled with 50 nM NHS-[3H]aminopterin, and
purified plasma membrane (20) from these cells was electrophoretically
fractionated by SDS-polyacrylamide gel electrophoresis using a 15%
polyacrylamide disc gel. The electrophoretic profile for radioactivity
shown in Fig. 3 delineates the major peak at about 58 kDa with some minor peaks discernible at a lower range of kDa. A
similar fractionation of NHS-[3H]aminopterin-labeled
plasma membrane from Fraction IV cells (not shown) did not delineate a
predominant peak within the same overall range of kDA. To associate the
differential in affinity labeling of Fraction I and Fraction IV cells
in the form of a 58-kDA protein with influx of
l,L-[3H]-5-CH3-H4folate
by Fraction I cells, we determined the effect of affinity labeling with
NHS-aminopterin on the influx of this folate compound. The data in Fig.
4 show that within the range of 0-10 µM,
affinity labeling with NHS-aminopterin effectively inhibited
(IC50 = 0.65 ± 0.05 µM) influx of
l,L-[3H]-5-CH3-H4folate.
It was also shown (data not given) that
l,L-5-CH3-H4folate and
folic acid were also effective inhibitors of
NHS-[3H]aminopterin labeling of Fraction I cells.
Detection of RFC-1 mRNA in Luminal Epithelium from Mouse Small Intestine
The data from a Northern blot in Fig. 5
show that a substantial level of RFC-1 RNA transcript was found with an
RFC-1 cDNA probe in this tissue compared with liver when both blots
were normalized to the level of -actin RNA transcripts in each
tissue. The direct detection of RFC-1 RNA in isolated Fraction I
versus Fraction IV cells by Northern blotting was not
possible because of the large number of Fraction IV cells that was
required for the preparation of adequate amounts of
poly(A)+ RNA. Because of the size of these preparations
(usually 20-30 mice each), the quality of the RNA was consistently
poor. Therefore, quantitative reverse transcription-PCR was used to
determine the relative amount of RFC-1 mRNA in preparations of much
smaller numbers of Fraction I and Fraction IV cells that were obtained from only a few mice. The results of these reverse transcription-PCR reactions in the form of a radioautograph are given in Fig.
6. The data shown are for the amount of RFC-1 cDNA
product obtained normalized to the amount of
-actin cDNA product
obtained when run in a parallel PCR reaction. From Fig. 6, it can be
seen that RFC-1 mRNA is present in Fraction IV cells at a much
lower level than in Fraction I cells.
Western Blotting
Further evidence for an association between
l,L-[3H]-5-CH3-H4folate
influx in Fraction I cells and RFC-1 gene expression was obtained in the form of immunoblotting with antibodies raised in
rabbits against anti-RFC-1-specific peptides. The blots in Fig.
7 reveal a prominent protein band at 58 kDa and a faint
band at 46 kDa in plasma membrane from Fraction I cells. In contrast, blotting of plasma membrane from Fraction IV cells revealed neither of
these protein bands but occasionally detected a weakly reacting protein
band in the vicinity of 35 kDa. Western blotting with anti-RFC-1
peptide antibody was also carried out with purified brush-border
membrane protein from Fraction I cells. From data shown in Fig.
6B, it can be seen that this antibody detected a 58-kDa
protein band in this specialized membrane in addition to another band
at ~35 kDa. A plot of plasma membrane protein from Fraction IV cells
run in parallel is also shown in Fig. 6.
Further Evidence for a Role of the Transporter Encoded by the RFC-1 Gene in Folate Absorption
The results of both
NHS-[3H]aminopterin affinity labeling and immunoblotting
with anti-RFC-1 peptide antibody presented above clearly suggest that
there is an association between a 58-kDa protein and folate transport
in absorptive luminal epithelium (Fraction I cells) from mouse small
intestine. More direct evidence was obtained by determining the effect
of the anti-RFC-1 peptide antibody on
l,L-[3H]-5-CH3-H4folate
influx by Fraction I cells at pH 6.2 in the presence of 150 µM DIDS. The results presented in Fig. 8
show that influx of this folate compound under these conditions by Fraction I cells was inhibited substantially by the prior incubation of
Fraction I cells with varying amounts of anti-RFC-1 peptide antibody
and that inhibition occurred in a concentration-dependent manner. No effect was obtained with an irrelevant antibody of the same
IgG class against a mouse folylpolyglutamate synthetase peptide
purified in the same manner from rabbit serum.
The studies described here on folate compound influx by isolated luminal epithelial cells from mouse small intestine are in close agreement with those published earlier (11, 13) by Selhub and Rosenberg using either everted sacs or brush-border membrane vesicles from rat small intestine. Under conditions that suppress non-pH-dependent influx (14), the role of a highly saturable, pH-dependent system in mediating influx of folate compounds that operates only in absorptive epithelial cells has been documented with characteristics very similar to that found (13) in the brush-border membrane of rat small intestine. These studies were further extended to show that the dependence of influx of l,L-[3H]-5-CH3-H4folate on pH reflected a difference at each pH in values for Vmax but not for apparent Km for this system. The basis for this effect on maximum capacity rather than saturability is not understood but could reflect a difference in the rate of translocation of the transporter in response to a difference in the proton gradient (inside versus outside) at each pH.
In further support of the notion that the acid pH-dependent system in the luminal epithelium of mouse small intestine is involved in folate absorption, we have also provided data that show that acid pH-dependent transport in luminal epithelium of this organ is developmentally regulated. Physiologically significant levels of acid pH-dependent influx of l,L-[3H]-5-CH3-H4folate and of the 58-kDa putative transporter occurs only in mature absorptive cells apparently within the brush-border membrane. The relevance of this protein to acid pH-dependent transport in these cells was strongly suggested by the results of folate-based affinity labeling and immunoblotting. Also, the murine RFC-1 gene, which encodes a 58-kDa membrane protein (15), appears primarily to be transcriptionally active in mature absorptive cells. Finally, it was found that the folate-based affinity label and antibodies against murine RFC-1-specific peptides will inhibit influx of l,L-[3H]-5-CH3-H4folate in mature absorptive cells.
In the report by Said et al. (16), the RFC-1 cDNA
derived from poly(A)+ RNA of mouse small intestine
incorporated a different 5 end from that initially reported for an
RFC-1 cDNA derived from L1210 cell poly(A)+ RNA. We
have now obtained data to show (31) that the RFC-1 cDNA derived
from mouse small intestine is a splice variant incorporating an
alternative to exon 1 of the RFC-1 gene, which encodes this different 5
end. Although supporting data are still needed, it is
likely that transcription of this splice variant, which is initiated at
the alternate to exon 1 in small intestine, is under the control of a
second promoter. Transcription of genes that encode alternate 5
ends
in the form of splice variants have frequently been shown (32-35) to
be under the control of multiple promoters. Additional work will be
required to establish this point.
Lastly, it is of interest to compare data obtained in the current studies on mediated folate compound transport in luminal epithelium of small intestine to that documented (1, 2, 12, 20, 36) in detail for L1210 cells. If we assume that transport of these compounds in both of these tissues is a direct result of RFC-1 gene expression alone, a paradox emerges pertaining to the properties of the transport system in each case. Although the murine RFC-1 gene encodes a 58-kDa protein, the mass of this protein actually detected is 58 kDa in luminal intestinal epithelium but only 46 kDa in L1210 cells (20, 36). Moreover, discrepancies exit with regard to both pH dependence and structural preferences among folate compounds as permeants. The transport system in L1210 cells exhibits (12) a moderately alkaline pH optimum and marked differences (1, 2, 12, 18) in preferences among folate compounds as permeants. In contrast, the system in mouse luminal intestinal epithelium exhibits (see above) an acid pH optimum and close similarity in preferences among the same folate compounds as permeants. There are a number of possible explanations for these apparent discrepancies that are consistent with the role of the RFC-1 gene in each case. These could pertain to alternate splicing, post-transcriptional modification and (or) the role of accessory proteins. However, since there is currently a lack of relevant data, further work will be required that addresses each aspect of this question.