Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois at Chicago and West Side Veterans Affairs Medical Center, Chicago, Illinois 60612
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ABSTRACT |
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The current studies were undertaken to
establish an in vitro cellular model to study the transport of
SO and hormonal regulation and
to define the possible function of the downregulated in adenoma
(DRA) gene. Utilizing a postconfluent Caco-2 cell line, we
studied the OH
gradient-driven
35SO
uptake. Our findings consistent with the presence of an apical carrier-mediated 35SO
exchange process in Caco-2 cells include: 1) demonstration
of saturation kinetics [Michaelis-Menten constant
(Km) of 0.2 ± 0.08 mM for
SO
1 · 2 min
1]; 2) sensitivity to inhibition by DIDS
(Ki = 0.9 ± 0.3 µM); and 3) competitive inhibition by oxalate and Cl
but not by nitrate and short chain fatty acids, with a higher Ki (5.95 ± 1 mM) for Cl
compared with oxalate (Ki = 0.2 ± 0.03 mM). Our results also suggested that the
SO
and
Cl
/OH
exchange processes in Caco-2 cells
are distinct based on the following: 1) the
SO
exchange was highly sensitive
to inhibition by DIDS compared with Cl
/OH
exchange activity (Ki for DIDS of 0.3 ± 0.1 mM); 2) Cl
competitively inhibited the
SO
exchange activity with a high
Ki compared with the Km
for SO
; 3) DIDS competitively inhibited the
Cl
/OH
exchange process, whereas it
inhibited the SO
exchange activity
in a mixed-type manner; and 4) utilizing the RNase
protection assay, our results showed that 24-h incubation with 100 nM
of thyroxine significantly decreased the relative abundance of
DRA mRNA along with the
SO
exchange activity but without
any change in Cl
/OH
exchange process. In
summary, these studies demonstrated the feasibility of utilizing Caco-2
cell line as a model to study the apical
SO
and
Cl
/OH
exchange processes in the human
intestine and indicated that the two transporters are distinct and that
DRA may be predominantly a SO
as well.
human intestine; anion exchangers; Cl/HCO
) exchange; SO
exchange
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INTRODUCTION |
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CONGENITAL CHLORIDE DIARRHEA
(CLD) is a rare autosomal disease characterized by a watery stool with
high chloride concentration and metabolic alkalosis (24).
Previous perfusion studies have demonstrated that the basic defect in
these patients is in the ileal and colonic luminal membrane
Cl/HCO
Previous studies from our laboratory, utilizing purified apical plasma
membrane vesicles, have demonstrated the presence of Cl/HCO
) exchange
process in the human colon (21). Additionally, we have
previously shown that AE2 and bAE3 but not AE1 and cAE3 are expressed
throughout the length of the human intestine (38). Our
immunoblotting studies demonstrated that the protein products of AE2
and bAE3 are localized to basolateral membranes of the epithelial cells
in all the regions of the human intestine (2). Recent
studies (39) from our laboratory have also demonstrated and characterized a sulfate/hydroxyl exchange process in the proximal colonic apical membrane vesicles and showed that this exchanger has
very low affinity for chloride compared with sulfate. The results of
these studies strongly suggested that the described sulfate/hydroxyl
exchange activity was distinct from the previously described
Cl
/HCO
) exchange
process. In light of the aforementioned, it is not yet clear whether
DRA protein product is in fact the apical anion exchanger
responsible for the Cl
/HCO
These intriguing findings warrant the development of a suitable in vitro model to study the sulfate and chloride uptake, their substrate specificity, and their regulation with various hormones and to examine the role of DRA in intestinal electrolyte transport. The colonic adenocarcinoma Caco-2 cell line has been shown to be a good model to study the mechanisms of electrolyte transport in the intestine (40). Additionally, thyroxine has previously been shown to alter the level of expression and the function of a number of electrolyte transporters in various tissues and cell lines (6, 8, 10). Therefore, our current studies were undertaken to examine whether Caco-2 cell line could serve as a suitable model to study the apical chloride and sulfate uptake mechanisms and their substrate specificity. Also, we examined the possible role of thyroxine in regulation of the expression of human DRA mRNA along with chloride and sulfate uptake activities in Caco-2 cells.
Our current studies demonstrated that Caco-2 cells could serve as an
experimental model to study the apical intestinal
SO and
Cl
/OH
exchange activities. Our data
demonstrated that the two transporters are distinct based on the
following: 1) in contrast to the
Cl
/OH
exchange process, the
SO
exchange activity was highly
sensitive to inhibition by DIDS (the AE inhibitor); 2) DIDS
competitively inhibited the Cl
/OH
exchange,
whereas it inhibited the SO
exchange in a mixed-type manner; 3) chloride inhibited the
SO
exchange competitively with a
high inhibition constant (Ki) for Cl
compared with the Michaelis-Menten consant
(Km) for SO
exchange activity, whereas the
Cl
/OH
exchange activity was not altered by
thyroxine. These data indicate that DRA may be primarily
responsible for sulfate transport although capable of transporting
chloride and that it appears to be distinct from the previously
described Cl
/HCO
)
exchanger in the intestinal luminal membrane.
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MATERIAL AND METHODS |
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Cell culture
Caco-2 cells were obtained from ATCC and cultured at 37°C in an atmosphere of 5% CO2. Cells were maintained as previously described (33) in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/l glucose, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 2 µg/ml gentamicin, 10 mM HEPES, 1% essential and nonessential amino acids, and 20% fetal bovine serum. For the uptake experiments, cells from passages between 20-25 were plated in 24-well plates at a density of 2 × 104 cell/ml. Confluent monolayers were then used for the transport experiments at day 10 postplating. To study the effect of 100 nM of thyroxine, we rendered cells quiescent by serum removal for 48 h before study. Control cells were treated with equivalent amounts of 100 nM NaOH (vehicle) to thyroxine-treated cells.35SO uptake
Designing of PCR primers and PCR technique
The PCR primer sequences for human DRA were designed from the human sequences that have been retrieved from the gene-bank CD-ROM utilizing GeneWorks software and as previously described (37). The primer sequences are 5' primer: ACCATGATTGAACCCTTTGGGAATCAGTAT; 3' primer: ATACACCTGCTGCAATCACG (length of amplified region 910 residues; nt 184-1094 of the human DRA). The PCR was essentially performed according to the manufacturer's instructions utilizing 2 µg of human colonic cDNA pool obtained from Invitrogen (Carlsbad, CA) as a template, gene-specific human DRA PCR primers, and the proofreading Elongase enzyme mix (GIBCO BRL, Gaithersburg, MD). The reaction was performed in a total volume of 50 µl of PCR mixture containing 60 mM Tris-SO4 (pH 9.1 at 25°C), 18 mM NH4SO4, 1.8 mM MgSO4, 200 µM each dNTPs, 400 nM of each primers, and 2 µl of the Elongase enzyme mix. The PCR was carried out using a Microcycler programmable heating/cooling dry block (Perkin Elmer, Norwalk, CT) for 40 cycles of amplification (94°C, 30 s; 52°C, 30 s; 68°C, 3 min) followed by 10 min at 68°C. PCR products were separated by electrophoresis on 1% agarose gel containing ethidium bromide (0.5 µg/ml). Bands of expected sizes were visualized under ultraviolet light utilizing Eagle eye II Still Video System (Stratagene, La Jolla, CA). The 910-bp PCR products were excised from the agarose, purified utilizing Sephaglas BandPrep Kit (Amersham Pharmacia Biotech, Piscataway, NJ), and subjected to A-tailing reaction by heating at 70°C for 30 min in a final volume of 10 µl containing 100 mM Tris · HCl (pH 8.3), 2.5 mM MgCl2, 50 mM KCl, 200 µM dATP, and 5 units of Taq DNA polymerase (GIBCO BRL). Two microliters of the reaction were ligated into a pGEM-T Easy vector (Promega, Madison, WI). The orientation and the sequence of the insert were confirmed by sequencing utilizing the Sequenase Kit (Amersham Pharmacia Biotech). The constructed DRA vector, p5'DRA, was then utilized for making cRNA probe for the RNase protection assay.Isolation of RNA
Total RNA was extracted from Caco-2 cells by the method of Chomczynski and Sacchi (7) using RNAzol solution supplied by the manufacturer (Tel-Test, Friendswood, TX) and essentially using the manufacturer's protocol.Generation of cRNA probes and RNase protection assay
p5'DRA vector was linearized by digestion with Ava II and transcribed with T7 RNA polymerase in the presence of [32P]CTP utilizing the riboprobe Gemini transcription system (Promega, Madison, WI). [32P]cRNA riboprobe for human DRA contained 579 bp, and the protected fragment corresponded to 527 bp. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) vector was constructed, and cRNA riboprobe for GAPDH was generated as described previously by us (11). [32P]cRNA for GAPDH contained 300 bp, and predicted protected fragment corresponded to 210 bp.For the RNase protection assay, total RNA (20-30 µg) was coprecipitated with 107 counts/min of high specific activity 32P-labeled DRA riboprobe and 105 counts/min of low specific activity 32P-labeled GAPDH riboprobe. Samples were then resuspended in a hybridization buffer containing 75% formamide, 400 mM NaCl, 1 mM EDTA, and 40 mM PIPES, pH 6.4, and were hybridized at 45°C for 12-18 h. Samples were then diluted in 10 vol of 300 mM NaCl, 5 mM EDTA, and 10 mM Tris pH 7.5, and 1,400 units of T1 ribonuclease were added to each sample. After a 45-min incubation at 37°C, samples were added to a stop solution containing 4 M LiCl and 5 µg tRNA and precipitated with 2 vol of ethanol. Precipitates were resuspended in a small volume of dye solution (xylene cyanole and bromophenol blue in 90% formamide and 10 mM EDTA, pH 7.5). The double-stranded 32P-cRNA fragments that were protected from the RNase digestion were heated at 95°C for 5 min and analyzed by electrophoresis on a denaturing polyacrylamide gel containing 8 M urea. The gels were dried and exposed to storage phosphor screen overnight and then analyzed utilizing a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The relative abundance of DRA was calculated by comparing the number of counts of radioactivity of DRA band in response to thyroxine treatment or vehicle alone after normalization to the number of counts in the GAPDH band. Because GAPDH mRNA is expressed in very high abundance compared with DRA, ~20-fold lower specific activity of 32P-labeled GAPDH compared with DRA probes were synthesized by including more cold CTP in the transcription reaction. This was specifically done to keep the density of GAPDH band in a readable range on final scan while enabling the detection of DRA band. The procedure of labeling was exactly the same every time, and once the probes were synthesized, the batch of probes was simultaneously utilized for hybridization with equal amounts of RNA.
Statistical analysis
Data are means ± SE of at least 3-6 independent determinations (performed in separate wells) repeated on at least two to three occasions. When error bars are not visible in the figures, they are smaller than the symbol. Statistical differences were analyzed by Student's t-test, and a P value of <0.05 was considered statistically significant. ![]() |
RESULTS |
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The OH gradient-stimulated
35SO
Time course of OH gradient-stimulated
35SO
gradient-dependent apical
35SO
gradient-driven
35SO
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Kinetics of SO
exchange process in Caco-2 cells.
To further characterize the apical
SO
exchange process in Caco-2
cells, we examined the kinetic parameters of the exchanger by measuring
sulfate uptake in the presence of increasing concentrations of
extracellular SO
gradient-stimulated
35SO
1 · 2 min
1. These data indicate a carrier-mediated process for
the OH
gradient-driven apical sulfate uptake in Caco-2
cells.
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35SO
exchange process was highly sensitive to inhibition by DIDS. To further examine the characteristics of the exchanger, we studied the effect of
DIDS on kinetic parameters of the apical
35SO
1 · 2 min
1. These experiments were repeated using different
concentrations of DIDS (0.015, 0.025, and 0.05 mM; not shown) and
demonstrated the same pattern of inhibition with a
Ki of 0.9 ± 0.3 µM.
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Effect of anions on 35SO/HCO
)
exchanger. Previously, DRA was shown to be a sulfate
and oxalate transporter. To examine the anion specificity of the apical
SO
exchanger in Caco-2 cells, we
investigated the effect of various anions on the sulfate uptake in
Caco-2 cells. As shown in Fig. 4, 5 mM
cis-concentration of butyrate, formate, lactate, succinate, and nitrate failed to significantly inhibit
35SO
antiporter in Caco-2 cells.
These results also indicate that the exchanger has a markedly higher
affinity for oxalate compared with chloride.
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The mechanism(s) of 35SO gradient-stimulated
35SO
exchanger could use oxalate and
chloride as alternative substrates in addition to sulfate, it appears
that the antiporter has a markedly higher affinity for both sulfate and
oxalate compared with chloride.
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OH gradient-stimulated 36Cl
uptake in Caco-2 cells
Time course of OH gradient-stimulated
36Cl
uptake.
We have previously characterized
Cl
/HCO
) exchange
process in the human colonic proximal and distal apical plasma membrane
vesicles. To investigate whether Caco-2 cells could also serve as a
model to study the chloride uptake, we examined the effect of outwardly
directed OH
gradient on the time course of
36Cl
uptake in Caco-2 cells. As shown in Fig.
7, OH
gradient-dependent
36Cl
uptake in Caco-2 cells was linear as a
function of time up to 7 min and it was inhibited by DIDS. It should be
noted that in contrast to SO
exchange activity, OH
gradient-stimulated
36Cl
uptake was much less sensitive to
inhibition by DIDS. These data suggest that in Caco-2 cells the two
processes may be occurring via two distinct transporters.
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The OH gradient-stimulated
36Cl
uptake inhibition by DIDS.
Because the SO
exchange process
was shown to be inhibited by DIDS in a mixed-type manner and Cl
/OH
exchange appeared to be more
resistant to inhibition by DIDS, we examined the mechanism of
Cl
/OH
exchange inhibition by DIDS in Caco-2
cells. As shown in Fig. 8, 0.35 mM DIDS
altered the apparent Km for chloride, whereas no
significant changes occurred in the Vmax. The
data also indicated a competitive mode of inhibition. These experiments
were repeated using different concentrations of DIDS (0.2, 0.5, and 1 mM; not shown) and demonstrated the same pattern of inhibition with a Ki of 0.3 ± 0.1 mM.
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The effect of thyroxine on OH gradient-stimulated
35SO
uptake in Caco-2 cells
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The effect of thyroxine on the relative abundance of human DRA mRNA
To investigate the possible mechanism of reduced SO
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DISCUSSION |
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In our current studies, we have established an in vitro cellular
model to study sulfate and chloride transport and have defined the
possible role of DRA in the anion transport in the human
intestine. Our results demonstrated that the Caco-2 cell line could
serve as a suitable model to study sulfate and chloride transport. The data of our studies demonstrated and characterized the presence of an
apical SO exchange process in
Caco-2 cells. The findings consistent with a carrier-mediated activity
for the OH
gradient-stimulated
35SO
exchange process, indicating
the presence of an anion antiporter that could use oxalate and chloride
as alternative substrates in addition to sulfate. Furthermore, the
results of the present studies indicated that the apical
SO
and
Cl
/OH
exchange in Caco-2 cells are distinct
processes based on the following: 1)
SO
exchange activity was more
sensitive to inhibition by DIDS compared with
Cl
/OH
exchange process; 2)
SO
exchange process was inhibited
by DIDS in a mixed-type manner, whereas
Cl
/OH
exchange was competitively inhibited
by DIDS; 3) incubation with 100 nM thyroxine inhibited the
OH
gradient-stimulated
35SO
uptake in Caco-2 cells; and
4) in parallel to reduced SO
Previous studies have shown that postconfluent differentiated Caco-2
cells possess many of the functional and structural characteristics of
the native enterocyte including similar transport mechanisms and
regulatory pathways (27). Therefore, Caco-2 cells have
been previously used as a model to characterize the electrolyte
transport in the small intestine and colon (40). Silberg
et al. (37) have recently shown that the DRA
gene is expressed in postconfluent but not preconfluent Caco-2 cells.
Consistent with the previous studies of Silberg et al., our results
showed that 5-7 days postconfluent but not preconfluent Caco-2
cells expressed the DRA gene (data not shown) and possess
apical SO and
Cl
/OH
exchange activities. Therefore, these
cells have been utilized here as an in vitro cellular model to study
the regulation of the DRA expression by thyroxine and
delineate the interactions between sulfate, chloride, and oxalate
transport in the human intestine.
The apical SO exchange in the
polarized monolayers of Caco-2 cells appeared to be a carrier-mediated process. The SO
exchanger in
Caco-2 cells exhibited kinetic characteristics similar to
SO
or
SO
exchanger in rabbit ileal BBM
(0.475 mM; Ref. 34),
SO
exchanger in Caco-2 cells
appears to be highly sensitive to DIDS. Our results showed that the
effect of DIDS on 35SO
exchange process needs more detailed characterization, this kind of
inhibition suggests that DIDS and SO
The apical SO exchange process in
Caco-2 cells appeared to be specific for SO
, and oxalate but not for the other anions such as
nitrate and short chain fatty acids. Our kinetic studies demonstrated
that both Cl
and oxalate inhibited the OH
gradient-stimulated 35SO
and oxalate suggest that although the
exchanger can transport Cl
and oxalate in addition to
SO
(Ki = 5.9 mM). In
agreement with the previous studies of Silberg et al.
(37), which demonstrated that DRA gene product
is a sulfate and oxalate transporter, our results indicate that
DRA may be responsible for the
SO
exchange process in Caco-2
cells. On the other hand, previous studies in the human proximal colon
and rabbit ileum (18, 21) have shown that nitrate and
bromide could substitute for chloride in the
Cl
/HCO
) exchange
processes. Because the SO
exchanger in the current study exhibited lower affinity for chloride and nitrate did not alter the OH
gradient-stimulated
35SO
and
Cl
/HCO
) exchange
activities in the human intestine may be distinct processes.
We have previously characterized a
Cl/HCO3
(OH
)
exchange process in the human small intestine and proximal colon (21,
29). These studies demonstrated that Cl
uptake into the
human colonic apical membrane vesicles was stimulated in the presence
of a pH gradient. The OH
gradient-stimulated
Cl
uptake into these vesicles was further stimulated in
the presence of a HCO
/HCO
/OH
exchange activities were mediated via
the same transporter. In the present study, we intended to examine
whether Caco-2 cells could also serve as a suitable model to study
chloride transport. Our data demonstrated the presence of an outwardly
OH
gradient-stimulated 36Cl
uptake that was linear as a function of time and could be inhibited by
DIDS. Because Caco-2 cells were derived from human colon adenocarcinoma and when confluent demonstrate the characterstics of differentiated enterocytes (27), it is most likely that the
OH
gradient-driven 36Cl
uptake
into these cells represents the activity of the same transporter that
is also responsible for Cl
/HCO
/OH
and
Cl
/HCO
uptake was not
(unlike our previous studies with human colonic apical membrane
vesicles) affected by imposing a HCO
/OH
but not the
Cl
/HCO
and
35SO
/HCO3
and
Cl
/OH
exchange processes with one that
could take 35SO
Similar to our previous findings in the human proximal colonic apical
membrane and to other intestinal AEs such as of rabbit and human ileum
(13, 18, 21, 29), the Cl/OH
exchange process in Caco-2 cells appeared to be relatively less sensitive to inhibition by DIDS. It has previously been suggested that
the possible explanation for the poor inhibition may be a result of
competition with the substrate (5). In agreement with
that, our findings showed that Cl
/OH
exchange process in Caco-2 is competitively inhibited by DIDS with a
Ki of 0.3 mM. The Ki
value for Cl
uptake inhibition by DIDS is also comparable
with other systems such as Cl
/HCO
and
Cl
/OH
exchange activities in Caco-2 cells
have different sensitivities and mechanisms of inhibition by DIDS with
different Ki values further supports the notion
that they are mediated via distinct transporters. Recent studies
(22, 23) have shown that DRA is capable of
transporting Cl
and suggested that DRA is the
intestinal apical Cl
/HCO
with a Ki of 5.9 mM indicating
that SO
exchanger is able to
transport Cl
but with a low affinity compared with
SO
The thyroid hormone thyroxine has been shown to have a widespread
effect on membrane transport of amino acids (12), glucose (35), and ions (6, 8, 10) in various tissues
and cell lines. For instance, Cano et al. (6) demonstrated
a stimulation of renal Na+/H+ exchanger by
transcripitional activation in OK cell line by thyroxine. Furthermore,
Chou et al. (8) have reported that the lysosomal sulfate
transport in the rat liver is decreased by thyroxine treatment. Additionally, thyroxine has been previously shown to play an important role in the developmental changes of a number of intestinal digestive enzymes and transporters (14). In this regard, Chow et al.
(10) have previously shown that thyroxine treatment to
suckling rats was followed by a 60% decrease in the expression of AE2
in the small intestine. The novel finding in the current study is that, in Caco-2 cells, 24-h treatment with thyroxine significantly decreased the OH gradient-driven sulfate but not chloride uptake.
This differential regulation indicates a specific effect of thyroxine
on the sulfate transporter and negates its generalized effect via
possible changes in membrane composition and fluidity as has been
observed under some circumstances (26, 32).
In the current studies, both the Vmax and the
Km for 35SO exchange process by
thyroxine. Consistent with the decrease in the
Vmax of sulfate uptake, our current results
demonstrated that thyroxine treatment in Caco-2 cells also
significantly decreased the relative abundance of DRA mRNA.
Because it has previously been demonstrated that the DRA
gene product is a sulfate and oxalate transporter that exhibits a high
homology to other sulfate but not chloride bicarbonate transporters, it
is most likely that in Caco-2 cells, DRA is responsible for
the pH-driven sulfate uptake that is regulated by thyroxine.
Additionally, our results suggest that, in Caco-2 cells, the
OH
gradient-driven chloride uptake occurs via a
transporter that is distinct from the DRA. In this regard,
Melvin et al. (22) have shown that mouse DRA
directly mediated the Cl
/HCO
/HCO
transport in the human colonic apical membranes;
3) the study did not provide evidence for the targeting of
the exogenous mouse DRA protein to the membrane of the
transfected cells; and 4) these studies did not rule out the
possibility of DRA functioning as part of a protein complex
that is responsible for the described Cl
/HCO
On the other hand, the findings of the current studies suggest that
chloride transport in Caco-2 cells is mediated by two distinct
transporters: 1) a SO
exchanger, which could be DRA, with ability to transport
Cl
as well but with low affinity; and 2) a
distinct Cl
/OH
exchanger. Previously, Kere
et al. (17) have proposed different models regarding
DRA and apical epithelial chloride transport. They suggested
that DRA might be contributing to the apical
Cl
/HCO
/HCO
/HCO
/HCO
) exchange
process, our results appear to support the last proposed model of Kere
et al. (17). In this model, a mutation in only one of the
proteins could block the functions of both transporters and cause the
observed defect in the Cl
/HCO
) exchange in CLD patients.
In conclusion, our current studies raise questions about the direct
role of DRA in intestinal chloride transport. Our results indicate for the first time that in Caco-2 cells the apical
Cl/OH
and
SO
exchange processes are distinct
and that DRA may be directly responsible for the
SO
exchange activity.
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ACKNOWLEDGEMENTS |
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These studies were supported by the Department of Veterans Affairs and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54016 (to P. K. Dudeja), DK-33349 (to K. Ramaswamy), and DK-09930 (to W. A. Alrefai).
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. K. Dudeja, Univ. of Illinois at Chicago, Medical Research Service (600/151), V. A. Medical Center, 820 South Damen Ave., Chicago, IL 60612 (E-mail: pkdudeja{at}uic.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 July 2000; accepted in final form 9 November 2000.
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