1 Department of Physiology and Pharmacology, Institute of Medical Biology, University of Southern Denmark-Odense University, Winsloewparken 21, DK-5000 Odense C; 2 Department of Physiology, University of Aarhus Ole Worms Alle 160, and 3 Department of Cell Biology, Institute of Anatomy, University of Aarhus University Park 233/234, DK-8000 Aarhus C, Denmark
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Inward Na+-HCO
cotransport has previously been demonstrated in acidified duodenal
epithelial cells, but the identity and localization of the mRNAs and
proteins involved have not been determined. The molecular expression
and localization of Na+-HCO
cotransporters (NBCs) were studied by RT-PCR, sequence analysis, and
immunohistochemistry. By fluorescence spectroscopy, the intracellular
pH (pHi) was recorded in suspensions of isolated murine
duodenal epithelial cells loaded with
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Proximal duodenal epithelial cells expressed mRNA encoding two electrogenic NBC1 isoforms and the electroneutral NBCn1. Both NBC1 and
NBCn1 were localized to the basolateral membrane of proximal duodenal
villus cells, whereas the crypt cells did not label with the anti-NBC
antibodies. DIDS or removal of extracellular Cl
increased
pHi, whereas an acidification was observed on removal of
Na+ or both Na+ and Cl
. The
effects of inhibitors and ionic dependence of acid/base transporters
were consistent with both inward and outward
Na+-HCO
cotransport. Hence, we propose
that NBCs are involved in both basolateral electroneutral
HCO
transport as well as basolateral electrogenic
HCO
transport in proximal duodenal villus cells.
intracellular pH; ion transport; fluorescence spectroscopy; sodium-bicarbonate cotransporter proteins
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
BICARBONATE SECRETION
FROM proximal duodenal epithelial cells is regarded as an
important protective factor against gastric acid (5) and
has been shown to include electrogenic as well as electroneutral
transport processes (34, 35, 40). These processes are
dependent on basolateral Na+ and are sensitive to
inhibition by the stilbene derivative DIDS, an inhibitor of various
Cl channels as well as most HCO
transporters (12). A model for proximal duodenal acid/base
transport was presented by Isenberg and coworkers (19):
luminal excretion of HCO
by
Cl
/HCO
exchange and an anion channel
was accompanied by basolateral Na+/H+
exchange and Na+-HCO
cotransport. The
basolateral transporters could be involved in both maintaining
intracellular pH (pHi) and in providing
HCO
for apical extrusion (also generated from
CO2 and H2O by action of the carbonic
anhydrase). Whether duodenal Na+-HCO
cotransport is electrogenic or electroneutral remains unknown, and the
basolateral localization of the transporter has not been verified. The
focus on this transporter has increased since the molecular cloning,
sequencing, and electrogenic characterization of the electrogenic renal
and pancreatic Na+-HCO
cotransporters
(NBC) (2, 30) and the electroneutral NBC (13,
27). Hence, the present study was undertaken to investigate the
molecular and functional expression of NBCs in duodenal epithelial
cells. We aimed to discriminate between electrogenic and electroneutral
NBC forms by molecular biological tools, to demonstrate the cellular
localization of the transporters, and to determine the direction of the
Na+-HCO
cotransport near steady-state
pHi in isolated cells.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. Amiloride hydrochloride, antibiotic-antimycotic solution (A9909, containing penicillin, streptomycin, and amphotericin), and RPMI 1640 medium (R7388) were obtained from Sigma Chemical (St. Louis, MO). 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was purchased from Molecular Probes (Eugene, OR). Primers for RT-PCR were synthesized by DNA-Technology. All other chemicals were of analytical grade. Drugs were dissolved in DMSO or ethanol, and final concentrations of DMSO or ethanol were below 0.1% (vol/vol), except for the RPMI medium at 0.4% (vol/vol). Antibodies directed against 15 amino acids of the COOH terminal of the electroneutral NBC from rat aorta (raNBCn1) or the electrogenic NBC1 from rat kidney (rkNBC1) were raised in rabbits (24, 39).
Cell preparation.
C57BL/6 mice weighing 16.7 ± 5.4 g were killed at 34 ± 7 days of age (mean ± SD; n = 28) by cervical
dislocation. After laparotomy, a 15-mm segment of proximal duodenum was
excised starting from 1 mm distal to the pylorus. The segment involved
the entire horizontal part of the murine duodenum (proximal to hepatic
and pancreatic outlets). Identical segments were used for functional
experiments and mRNA extraction. The lumen was rinsed and exposed to a
citrate solution for 5 min to remove the mucous layer. Final
concentrations were (in mM): 134.2 Na+, 9.5 K+,
97.5 Cl, 5.6 HPO
, 8.0 H2PO
, 27.0 citrate, and 10 glucose. pH
was adjusted to 7.4, and 5.6 µM indomethacin was added. The luminal
surface was then exposed to the EDTA-containing solution for 20 min at
37°C. Final concentrations (in mM) were: 153.3 Na+, 4.7 K+, 140.2 Cl
, 8.2 HPO
,
1.5 H2PO
, 1.0 EDTA, and 10 glucose. pH
was adjusted to 7.4, and 5.6 µM indomethacin was added. Indomethacin
was present during the cell isolation to prevent stimulation of
HCO
secretion secondary to release of prostaglandin
caused by the manipulation of the duodenum. Epithelial cells were
separated from the structural components of the duodenum by gentle
manipulation as described earlier (26).
Design of PCR primers.
To identify highly conserved regions of the
Na+-HCO cotransporters, we aligned
sequences of known electrogenic Na+-HCO
cotransporters from salamander kidney NBC (GenBank accession no.
AF001958) (30), rat renal NBC (AF004017)
(29), mouse pancreatic NBC (AF020195), and human kidney
NBC (AF007216) (11). Primers were constructed to recognize
either conserved regions of the electrogenic NBC types or regions that
were specific to the renal or pancreatic type, respectively. For
detection of an electroneutral NBC, primers were constructed on
similarity between rat electroneutral NBCn1 (AF070475)
(13) and human electroneutral NBC3 (AF047033) (27). Table 1 shows the
various primer sequences. Start base numbers correspond to those of rat
renal NBC1, mouse pancreatic NBC1, or rat electroneutral NBCn1.
|
RT-PCR and sequence analysis. Isolated duodenal epithelial cells were lysed, and total RNA was extracted using the RNeasy mini kit (Qiagen). RNA (125 µg/ml) was denatured in the presence of 62.5 µg/ml oligo(dT) primer (GIBCO BRL) and 187.5 µg/ml yeast tRNA at 95°C for 3 min in a volume of 8 µl. Denatured RNA was reverse transcribed by incubation with (final concentrations): 0.5 mM (of each base) deoxyribonucleotides (Pharmacia), 10 mM dithiothreitol, 1 IU/µl RNasin (Promega), 0.5 µg/µl BSA, 10 U/µl RT (GIBCO BRL), and buffer in a total volume of 20 µl for 60 min at 37°C. The reaction was terminated by heating at 95°C for 4 min.
The cDNA product was amplified by PCR. The following was added to 3 µl of cDNA produced by the RT reaction (final concentrations): 250 µM (of each base) deoxyribonucleotides, 50 U/ml Taq polymerase (Boehringer Mannheim), and 0.5 µM of each of the two primers, PCR buffer, and water to a final volume of 20 µl. After a denaturation at 95°C for 3 min, 30 cycles of PCR amplification were performed: denaturation at 95°C for 30 s, annealing at 52-60°C (dependent on primer specifications) for 30 s, and polymerization at 72°C for 30 s. The last cycle included 10 min at 72°C. A 15-µl sample of the PCR product and loading buffer was analyzed by electrophoresis on a 2% agarose gel. The gel was stained with ethidium bromide and photographed under ultraviolet illumination. Negative controls were yeast tRNA without duodenal RNA in the RT reaction and PCR and the omission of RT in RNA-containing samples during the RT reaction. RNAs from kidney, pancreas, or aorta extracts were used as positive controls for the renal and pancreatic NBC1 isoforms or the electroneutral NBCn1, respectively. Bands of predicted molecular weight for each PCR product were excised from the gel and purified using the QIAquick gel extraction kit (Qiagen). Both strands were sequenced using the corresponding specific primers and analyzed on a 310 genetic analyzer (ABI PRISM; PE Applied Biosystems).Immunohistochemistry. Duodenum from normal NMR1 mice were fixed by perfusion via the right heart ventricle with 2% paraformaldehyde in 0.1 M cacodylat buffer (pH 7.4). Blocks were dehydrated and embedded in paraffin. For light- and laser-confocal microscopy, the paraffin-embedded proximal duodena were cut at 2 µm on a rotary microtome (Micron). The staining was carried out using indirect immunofluorescence or indirect immunoperoxidase. The sections were dewaxed and rehydrated. On the sections for immunoperoxidase, endogenous peroxidase were blocked by 0.5% H2O2 in absolute methanol for 10 min at room temperature. To reveal antigen, we pretreated the sections by boiling them in 1 mM Tris (pH 9) supplemented with 0.5 mM EGTA using a microwave oven. Nonspecific binding of immunoglobulin was quenched by incubating the sections in 50 mM NH4Cl for 30 min, followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with relevant antibodies diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. After a wash in PBS supplemented with 0.1% BSA, 0.05% saponin, and 0.2% gelatin for 3 × 10 min, the sections for laser confocal microscopy were incubated in Alexa 488-conjugated goat anti-rabbit antibody (Molecular Probes) diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100 for 60 min. After a wash in PBS for 3 × 10 min, the sections were mounted in glycerol supplemented with antifade reagent (n-propyl gallate). For immunoperoxidase, the sections were washed, followed by incubation in horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (DAKO P448) diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. The peroxidase activity was visualized by 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Kem En Tek) dissolved in PBS supplemented with 0.1% H2O2. Counterstaining was carried out using Mayer's hematoxylin. Finally, the sections were dehydrated in graded alcohol and mounted in hydrophobic mounting medium (Eukitt). The microscopy was carried out using a Leica DMRE light microscope and a Zeiss LSM510 laser confocal microscope.
Determination of pHi.
The recording of pHi was performed by cuvette-based
spectroscopy using the membrane-permeant form of the pH-sensitive
fluorescent dye BCECF-AM as previously described (26).
Aliquots (200 µl) of cell suspension in RPMI were kept at 2°C until
use. The RPMI medium was adjusted to 1.0 mM Ca2+, 1 mg/ml
albumin, 5.6 µM indomethacin, 5 IU/ml penicillin, 5 µg/ml
streptomycin, and 12.5 ng/ml amphotericin B. Antibiotics/antimycotics were added to prevent microbial growth before and during dye loading. The cells were transferred to 37°C for 15 min, and dye was loaded in
5 µM of BCECF-AM for an additional 15 min and then washed. Cuvettes
containing 1.8 ml of cell suspension were placed in a dark chamber at
37°C, provided with H2O-saturated 95% O2-5%
CO2 under an air-tight cap, and continuously stirred. The
emitted light at 535 nm was recorded, and a ratio was calculated for
excitations at 490 and 440 nm. The fluorescence ratio (490/440) was
calibrated to pHi by the high K+/nigericin
technique (37). The data points were fitted to a third-order polynomial function: pHi(x) = 4.1702 + 5.1816 × x 3.4078 × x2 + 1.002 × x3. The total buffering capacity
(
tot) was determined from the calculated contribution
from the HCO
/CO2 buffer system
(10), and the intrinsic buffering capacity was obtained previously (26) involving stepwise decreasing
NH
concentrations. The equation was
tot= [2.3 × 9.6 × 10
7/10
pHi] + [186.1
23.5 × pHi]. The data from the experiments are presented as
pHi vs. time traces. Mean values of pHi were
determined from the inverse exponential fit of the individual traces
before and after the medium was changed. At predefined levels of
pHi, the slope of the fitted recovery curve was determined
(
pHi/dt). The net H+ efflux at
the given pHi was calculated by multiplication of the
pHi/dt with the buffering capacity for the
pHi value. The light emission was quenched in recordings
from samples containing amiloride or DIDS. Therefore, these samples
were calibrated in the presence of the inhibitors. Data are presented
as means ± SD. The Mann-Whitney rank-sum test was used for
statistics, and values of P < 0.05 were considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Detection of NBC forms by RT-PCR and sequence analysis.
RT-PCR was performed with mRNA from duodenal epithelial cells to study
the molecular expression of NBCs. Initially, RT-PCR was performed to
detect a nucleotide fragment that was conserved in the renal and the
pancreatic type electrogenic NBC1. As illustrated in Fig.
1A, we detected a band of the
expected molecular size of 760 bp in both duodenum and the control
tissue (kidney extract). The nucleotide sequence of the duodenal PCR
product shared 99.3% identity with mouse pancreatic NBC (AF020195)
corresponding to 100% homology of the predicted amino acid sequences.
However, a similar homology of the predicted amino acid sequence was
also found to rat renal NBC (AF004017) based on 96.3% nucleotide identity. The band was detected only when reverse transcription was
performed (Fig. 1A). It should be noted that, besides the band of expected molecular size, a weaker band appears (lane
6). This is also true for several lanes of the following RT-PCR
figures. The nucleotide sequence analysis was performed only on bands
of predicted length. To discriminate between the renal and pancreatic type NBCs, primers were constructed to recognize each of the two NH2 terminal sequences. The complete coding region of each
of the NH2 terminals was covered by one of the specific
sense primer and a common antisense primer located in the conserved
region of the sequence. As illustrated, bands were detected for the
renal type NBC (209 bp; Fig. 1B) as well as for the
pancreatic type NBC (341 bp; Fig. 1C) using duodenal cDNA.
Similar bands were obtained with the kidney and pancreas control lanes
(Fig. 1, B and C). The sequences of both duodenal
PCR products were then determined. The renal type NBC NH2
terminal fragment obtained from duodenal cDNA shared 98.9% nucleotide
identity with rat renal electrogenic NBC, yielding 100% homology of
the predicted amino acid sequences. The pancreatic type NBC
NH2 terminal from duodenal cDNA shared 98.0% nucleotide
identity with mouse pancreatic electrogenic NBC, amounting to 100%
homology of the predicted amino acid sequences. In Fig. 1C,
-actin bands were detected of a molecular size of ~206 bp. The use
of a
-actin primer pair as positive control for the presence of cDNA
also served as a marker for the purity of the mRNA used for reverse
transcription. The primers are located on each side of an intron in the
genomic sequence. PCR products of ~270 bp are obtained if the
reverse-transcribed mRNA (cDNA) is contaminated by chromosomal DNA. The
code for the transmembrane domain and COOH terminal of mouse duodenal
NBC1 was then sequenced by multiple RT-PCR reactions using primer pairs
designed on the basis of homology between mouse and rat electrogenic
NBC isoforms (Table 1). By use of the sequence for the renal type
NH2 terminal, the complete coding region of the murine
renal type NBC1 was a 3,035-bp sequence sharing 94.5% nucleotide
identity with rat renal electrogenic NBC, amounting to 98.6% homology
of the predicted amino acid sequences. The complete mouse renal type
NBC was given GenBank accession no. AF141934. By use of the sequence
for the pancreatic type NH2 terminal, the complete coding
region shared 94.9% nucleotide identity with mouse pancreatic NBC,
amounting to 98.4% homology of the predicted amino acid sequences. The
recent cloning and sequencing of the electroneutral NBCn1 provided the opportunity to study the expression of mRNA encoding this protein. As
shown in Fig. 1D, a band of the predicted molecular size of 362 bp was detected with duodenal epithelial cell cDNA. A similar band
was detected using rat aorta cDNA as positive control.
-Actin bands
were detected of a molecular size of ~206 bp. The duodenal fragment
of the electroneutral NBCn1 shared 96.0% nucleotide identity with rat
NBCn1 and 100% amino acid homology. The mouse NBCn1 fragment was given
the Genbank accession no. AF218295.
|
Immunolocalization of NBC isoforms.
Staining of the basolateral membranes of proximal duodenal villus cells
was obtained with the rabbit anti-NBCn1 antibody as shown in Fig.
2, A and
C. The rabbit anti-rkNBC1 antibody also stained the
basolateral membranes of villus cells from mouse proximal duodenum
(Fig. 2, B and D). The crypt cells were not
labeled with the anti-NBCn1 antibody (Fig. 2E) or by the
anti-rkNBC1 antibody (Fig. 2F). The basolateral labeling
found with both antibodies was confirmed by laser confocal images using
immunofluorescence (Fig. 2, G and H). For both
antibodies, the labeling was completely prevented by omission of
primary antibody and by preadsorption with 0.2 mg/ml of the relevant
peptide (performed with both peroxydase and fluorescence labeling; not
shown). It is noted that the anti-rkNBC1 antibody labeled the apical
and not the basolateral membrane in initial experiments performed
without perfusion fixation of the tissue or microwave antigen retrieval
(not shown).
|
Influence of acid/base transport inhibitors and removal of
extracellular Cl or Na+ on
pHi.
The function of NBCs was initially investigated by pHi
recordings near steady-state pHi. In Fig.
3 A-E, the recording was initiated as the medium was switched from HEPES-buffered salt solution
(solution 1, Table 2) to a
CO2/HCO
-HEPES-buffered salt solution
(solution 2). After 5 min, the composition of the medium was
changed either by addition of inhibitors (3.6-7.2 µl of
inhibitor in DMSO to a 1.8-ml sample followed by gentle mixing) or by
centrifugation and resuspension in Na+- and/or
Cl
-depleted media. The pHi was determined
after 5 min and after 10 min of measurement, i.e., after 5 min exposure
to CO2/HCO
and 5 min after the change of
medium. The net H+ efflux was calculated at the last second
before and at the first second after the change of medium. Initially,
the effects of inhibitors of HCO
transporters (200 µM DIDS) or Na+/H+ exchange (1 mM amiloride)
were studied. As illustrated in Fig. 3, A and F,
addition of DIDS effectively increased both pHi and the net
H+ efflux (both P < 0.05;
n = 5). In contrast, amiloride had only a minor effect
on both pHi [not significant (ns)] and net H+
efflux (P < 0.05; n = 5). Next, the
dependence on extracellular Na+ and Cl
was
studied (Fig. 3, B and F). Removal of
extracellular Cl
caused an increase of the net
H+ efflux (P < 0.05; n = 5), resulting in a slight elevation of pHi (statistically
significant only if values before and after manipulation were day
matched). Na+ removal or removal of both Na+
and Cl
caused a decrease of pHi and negative
net H+ efflux values (all P < 0.05;
n = 5). Accordingly, acid extrusion and/or alkaline
uptake were Na+ dependent, and a component of alkaline
extrusion was Cl
dependent. The pHi decrease
in Na+-free medium is largely independent of extracellular
Cl
.
|
|
|
Recovery of pHi after acidification or alkalization.
The pHi regulation was then studied in a broader pH range
by inducing an intracellular acidification by a prepulse of 30 mM NH. CO2/HCO
were
present only during pHi recovery to increase the
probability of an inward HCO
gradient and to obtain
a more pronounced acidification. Figure
5, A and B, shows
the pHi dependence of the net acid extrusion of acidified
duodenal epithelial cells. The traces of the recovery period were
fitted to inverse exponential functions, and the mean values for
H+ efflux were obtained from the curve fit at different
values of pHi. For all levels of pHi, the net
H+ efflux was reduced by removal of Na+ and by
addition of 1 mM amiloride or a high concentration of DIDS (500 µM).
The reduction of the H+ efflux was greater when
Na+ was omitted than when amiloride or DIDS was added for
all values of pHi (P < 0.05 in the
pHi range from 6.7 to 6.95). The amiloride-sensitive component of the H+ efflux (difference between control and
amiloride curves) was larger at low pHi, whereas the
DIDS-sensitive component (difference between control and DIDS curves)
was rather uniform within the pHi interval studied. The
Na+-dependent H+ efflux amounted to 85.4 ± 1.0% of control at pHi 6.80 (P < 0.05; n = 4), whereas the amiloride-sensitive and the
DIDS-sensitive H+ efflux at pHi 6.80 comprised
68.5 ± 2.3 and 25.6 ± 10.1% of control H+
efflux, respectively (for both P < 0.05;
n = 4). Hence, the recovery from acid pHi
contains both DIDS- and amiloride-sensitive components. The difference
between the amiloride-insensitive flux and the Na+-independent flux is larger than that observed in the
absence of CO2/HCO
(not shown). This
indicates the participation of Na+-HCO
cotransport.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we demonstrated the expression of multiple NBCs in
isolated epithelial cells from proximal mouse duodenum. The molecular
expression of the NBC forms was studied by RT-PCR, and the localization
was studied by immunohistochemistry. Inward Na+-HCO cotransport was identified as
the Na+-dependent, Cl
-independent, and
relatively DIDS-sensitive alkaline uptake in the presence of
CO2/HCO
. Outward
Na+-HCO
cotransport was suggested by
finding a more DIDS-sensitive and Cl
-independent alkaline
extrusion in the presence of CO2/HCO
.
Although the functional presence of
Na+-HCO cotransport has been
demonstrated in acidified duodenal epithelial cells from rat and humans
(4, 19), the molecular nature of the transporter has not
yet been identified. The recent cloning and sequencing of many NBC
forms made further analysis of duodenal Na+-HCO
cotransport possible. The RT-PCR
experiments and sequence analysis demonstrate the expression of at
least two putative electrogenic NBC1 isoforms by use of mRNA from
isolated duodenal epithelial cells. This is the first evidence for the presence of electrogenic NBCs in this tissue. The next step
was to discriminate between the pancreatic and the renal types of the
electrogenic NBC1, which differ only in the NH2 terminal. We expected to identify the pancreatic-type NBC1, because both the
duodenal epithelium and the pancreatic ducts secrete a large amount of
HCO
into the lumen of the respective organs.
Nevertheless, mRNA encoding both the pancreatic and renal isoforms of
the NBC1 was detected in the preparation.
The murine sequence of the renal-type NBC1 has not previously been determined. Given the importance of the murine model in membrane transport research, we sequenced the complete coding region of the cDNA. We did not find complete identity to the published mouse pancreas NBC1 in the transmembrane segment and COOH terminal of the coding sequence. Whether this could be explained by genetic differences between the mouse strains is unknown. It is noted that the predicted amino acid sequence encoding the consensus sites for DIDS binding of anion exchanger (AE) and NBC1 isoforms was conserved in the mouse renal-type NBC1: at predicted amino acids 559-562 a KMIK sequence is found, and at amino acid 768-771 a KLKK sequence is located.
The expression of mRNA encoding NBCn1 suggests that the duodenal
epithelial cells are also capable of electroneutral
Na+-HCO cotransport. This novel
detection of NBCn1 expression in the gastrointestinal tract adds to the
growing list of epithelia expressing this NBC protein, e.g., the
testes, lung, and kidney (13). However, the expression of
at least three NBC forms in the preparation should give rise to major
concern regarding the quality of the mRNA. Arguments against the
occurrence of false-positive PCR products are described in the
following. First, the actin primers were applied in RT-PCR experiments
to detect contamination with chromosomal DNA. As described in
RESULTS, the primers covered an intron-spanning segment of
the actin gene, and on contamination the band would be detected at
higher molecular weight than observed (~270 bp). Second, the
brightest bands of the PCR were always of the predicted size, although
additional bands occurred in several lanes. However, the sequence
analysis provided strong evidence for the specificity of the bands with
very high homology to known sequences from other species. Third, a
control sample of mRNA without RT was included in the first RT-PCR
experiment after each isolation of mRNA. This was performed to test the
purity of the mRNA both from duodenal epithelial cells and for the
control tissue. Chromosomal DNA, if present, would have caused the
formation of PCR bands in these controls. Finally, no introns were
detected during the sequencing of the RT-PCR products of mouse NBC
forms, including the complete coding region of the murine renal-type NBC1. Different physiological functions of the NBC proteins may then be
expected within the epithelial cells or along the crypt-villus axis.
The epithelial cells of duodenum include numerous typical villus
enterocytes (~80%), fewer crypt cells (<20%), and a few percent
goblet, paneth, and enteroendocrine cells (25). Expression of one of the NBC forms in, for example, the goblet cells or endocrine cells could provide the observed result because of the high sensitivity of the method. Hence, immunohistochemical staining of the duodenum was
conducted to confirm the expression of the NBC forms at protein levels
and to determine its localization within the epithelium.
The immunohistochemical localization of the NBCn1 protein on the
basolateral membrane of villus cells suggests a role of this transporter in cellular uptake of HCO from the
interstitial fluid. The electroneutral properties of NBCn1 (1,
13) or NBC3 (27), along with a presumed inward
Na+ gradient, predict a net influx of Na+ and
HCO
by the protein. This influx could either serve
as a basolateral donor of HCO
for apical secretion
of the ion or simply function as a regulatory system to maintain a
suitable pHi. The basolateral localization of the
electrogenic NBC1 protein was expected, since
Na+-HCO
cotransport is believed to occur
only at the basolateral membrane of duodenal epithelial cells
(19). The renal NBC1 is localized to the basolateral
membrane of the proximal tubule cells from the kidney (31)
and extrudes Na+ and HCO
(9). On the other hand, the pancreatic Na+-HCO
cotransport appears to mediate
net influx of the ions (20, 21) but is also localized to
the basolateral membrane (23). In any case, the expression
of an electrogenic NBC1 protein in the basolateral membrane introduces
the possibility that this NBC contributes to the electrogenic alkaline
transport across the epithelium of proximal duodenum. Interestingly, it seems that both electrogenic and electroneutral NBC forms are expressed
in the majority of villus cells rather than being confined to different
cell types within the epithelium. It should be noted that the rkNBC1
antibody was directed against amino acids common to both the pancreatic
and renal NBC1. The similar binding pattern of the rkNBC1 and the
raNBCn1 antibodies was not caused by cross-reactivity of the
antibodies, since they were directed against the very dissimilar NH2 terminals of the proteins. Furthermore, the antibodies
have been used to show differential localization of the electroneutral and the electrogenic NBCs in the kidney (24, 39).
The function of NBCs in duodenal epithelial cells was then assessed by
measurements of pHi. The demonstration of different modes
of Na+-HCO cotransport in duodenal
epithelial cells seems very complicated, since multiple other acid/base
transporters are expressed in the cells. Nevertheless, the
contributions of the different transporters to near-steady-state
pHi were investigated on the basis of their respective
sensitivity toward inhibitors and their ionic requirement. In the model
for duodenal acid/base transport presented by Isenberg and coworkers
(36), basolateral inward
Na+-HCO
cotransport and
Na+/H+ exchange are counteracted by apical
Cl
/HCO
exchange and, presumably, an
anion channel. Both the Na+-HCO
cotransport and Cl
/HCO
exchange seem
to be relatively DIDS insensitive, as judged from the previously
applied high concentrations of stilbene derivative (4,
19).
The detection of alkaline transport by various HCO
transporters depends on minimal acid extrusion by the Na+/H+ exchangers near steady state in the
presence of CO2/HCO
. In the present
study, duodenal Na+/H+ exchange was minimal
above pH 7.3, since the inhibitor amiloride had little effect
on pHi near steady state in the present study. This is
consistent with previous observations performed in the absence of
CO2/HCO
(26). The removal
of Na+ provided the pHi decrease observed
earlier (4, 19). This acidification is most likely to be
caused by outward Na+-HCO
cotransport
and reversed by Na+/H+ exchange, since it was
independent of extracellular Cl
, DIDS sensitive, and
partly inhibited by amiloride.
It was surprising that DIDS greatly increased pHi by
inhibition of alkaline extrusion at a relatively low concentration (200 µM). It should be emphasized that the pHi increase
induced by DIDS was much larger than that observed with
Cl removal. This suggests the involvement of a
DIDS-sensitive HCO
extruder different from the AE,
since Cl
/HCO
exchange by the AE2
isoform, which is present in duodenum, is about five times less DIDS
sensitive than AE1 (16). Given the expression of several
NBC forms in the duodenum, we hypothesized that the increase of
pHi might be due to inhibition of alkaline extrusion
through a more DIDS-sensitive NBC. The induced pHi increase
then arises from unopposed acid extrusion and/or alkaline uptake. Under
the present conditions, this is likely to be inward, relatively
DIDS-insensitive Na+-HCO
cotransport
given the low rate of Na+/H+ exchange at the
actual pHi. Interestingly, DIDS has been used in
concentrations of 50-400 µM to block electrogenic
Na+-HCO
cotransport (2, 10, 14,
30), whereas concentrations of 500 µM-1 mM of DIDS, or
its analogs, induced incomplete inhibition of
Na+-HCO
cotransport by electroneutral
NBC (13, 27) or Na+-HCO
cotransport in acidified duodenal epithelial cells (3, 4).
The apparent differences in sensitivity among the NBC forms toward DIDS
are mirrored by the observation that the electroneutral NBC lacks one
of the two putative DIDS-binding motifs that characterize the
electrogenic NBC1 (13, 27, 30).
The net alkaline uptake induced by DIDS was present both when applied
in normal medium and when Cl was removed. This would not
be the case if the Cl
/HCO
exchanger
was the only DIDS-sensitive alkaline extruder, since this transporter
would already be inhibited or reversed by Cl
removal.
Furthermore, the effect of DIDS was actually greatest in
Cl
-free medium, suggesting additive effects of
Cl
/HCO
exchanger inhibition/reversal
and the inhibition of an additional DIDS-sensitive alkaline extruder. Interestingly, the alkalizing effect of DIDS was conserved in relatively Cl
-depleted cells in the continued absence of
extracellular Cl
. Under these conditions,
Cl
/HCO
exchange in both directions
should be greatly minimized. This finding also excludes the
participation of Na+-dependent
Cl
/HCO
exchange and makes the
participation of an anion channel less likely.
Theoretically, the observed alkaline extrusion could be mediated by
other HCO transporters than outward Na+-HCO
cotransport. The cystic fibrosis
transmembrane conductance regulator (CFTR) has recently been
proposed to mediate HCO
extrusion in duodenum
(15, 17, 32). In the current study, this anion channel is
unlikely to contribute significantly to pHi regulation,
since CFTR was found to be expressed predominantly in duodenal crypt
cells (6, 17, 38) and a subpopulation of villus cells that
comprises a minor fraction of the total epithelial cell population
(6, 7). Furthermore, CFTR is inhibited by DIDS only by
intracellular application (18, 22). The subject of the
present study was restricted to acid/base transport in unstimulated
duodenal epithelial cells. Accordingly, the involvement of CFTR in
cAMP-stimulated duodenal HCO
extrusion was beyond
the scope of this study. If significant alkaline extrusion occurred
through another relatively nonselective anion channel, the transport of
HCO
through this channel should be increased under
Cl
-free conditions (on both sides of the plasma
membrane). Such an increase of alkaline extrusion was not observed in
the relative Cl
-depleted cells. Until this point, the
findings are consistent with the presence of outward-directed
Na+-HCO
cotransport in addition to the
previously described acid/base transporters:
Cl
/HCO
exchange,
Na+/H+ exchange, and inward
Na+-HCO
cotransport. It should be noted
that the degree of Cl
depletion is uncertain, because the
intracellular Cl
concentration was not determined.
Pretreatment in Cl
-free solutions has been reported to
induce significant reductions of intracellular Cl
levels
in several cells types (8, 33). It can be hypothesized that membrane potential-insensitive alkaline uptake (presumably by
NBCn1) is favored at low pHi, only to be counteracted by
alkaline extrusion near steady-state pHi by a membrane
potential-sensitive process (presumably NBC1). Indeed, the net
H+ efflux was not affected by 50 mM K+ in
acidified cells, whereas the net H+ efflux was increased at
pHi 7.25 and the pHi after 5 min was slightly
elevated by 50 mM K+. This supports to some extent the
involvement of a membrane potential-sensitive component of duodenal
HCO
transport and is thus consistent with the
molecular biological demonstration and the immunohistochemistry.
Nevertheless, more direct measurements are required to confirm this finding.
The second approach to the study of the duodenal acid/base transporters
was to remove the pHi from steady-state values and study
the recovery of pHi. The pHi recovery of
acidified cells was strongly inhibited by the absence of extracellular
Na+, consistent with previous demonstration of
Na+/H+ exchange and inward
Na+-HCO cotransport (19).
The fact that only a minor part of the pHi recovery is
Na+ independent largely excludes the presence of other
major acid extruders of alkaline uptake systems in the cells. The net
H+ efflux was sensitive to a high concentration of DIDS
(500 µM) in the entire pHi range. However, DIDS-sensitive
H+ efflux seems to be constant from pHi 6.7 to
7.0, implying that the inward Na+-HCO
cotransport is relatively insensitive to pHi in this range.
Higher concentrations of DIDS were avoided because of massive and
varying quenching of the signal by the compound. In contrast, the
amiloride-sensitive component was strongly pHi sensitive,
as reported previously (26), and seemed to account for the largest fraction of the pHi recovery at low
pHi values. This
Na+/H+exchange-mediated H+ efflux
might be slightly underestimated, since the relatively amiloride-resistant Na+/H+ exchange isoform
NHE3 is present in duodenal epithelial cells (26). The
similar net H+ efflux from acidified control cells and
relatively Cl
-depleted epithelial cells supports the
Cl
-independent nature of the alkaline uptake observed
without cellular acidification.
The outward HCO transport was studied in cells
alkalized by propionic acid prepulse. The pHi recovery of alkalized cells was strongly dependent on extracellular
Cl
and was probably mediated by
Cl
/HCO
exchange. The minimal net
OH
efflux in the absence of Cl
could,
however, arise from another outward HCO
transport,
slightly exceeding an ongoing inward
Na+-HCO
cotransport. We observed the
highest rate of pHi recovery in the absence of
extracellular Na+. In support of this view, full recovery
of pHi was observed only in the absence of extracellular
Na+. This seems consistent with a facilitation
of outward HCO
transport because inward
Na+-HCO
cotransport was prevented by
Na+ removal. Whether such outward HCO
transport is mediated by Na+-HCO
cotransport or by a HCO
channel could not be determined.
Taking the molecular biological and the functional findings together,
it seems likely that basolateral localized electroneutral NBCn1
mediates the relatively DIDS-insensitive inward
Na+-HCO cotransport in villus cells, as
illustrated in the modified model of duodenal acid/base transport (Fig.
7). This transport should be driven by an
inward-directed chemical Na+ gradient and be independent of
the electric gradient of the ions across the plasma membrane. It is
suggested that basolateral electrogenic NBC1 forms are either involved
in inward Na+-HCO
cotransport or mediate
the more DIDS-sensitive outward transport of HCO
presently demonstrated in isolated proximal duodenal epithelial cells.
The electrogenic NBC isoforms are believed to transport two or three
molecules of HCO
per Na+, as recently
reviewed (28). Outward HCO
transport
would be possible despite the inward-directed electrochemical gradient
for Na+, assuming an apical transmembrane potential around
70 mV, a 3:1 stoichiometry, and a typical ionic distribution across
the plasma membrane. Such outward electrogenic
Na+-HCO
cotransport could, however,
result from the nonpolarized state of the cells during measurements.
The actual ionic distribution and the membrane potential of the
isolated cells are not known, and the direction of transporter in situ could be either inward or outward. In conclusion, evidence is provided
for the expression of both electrogenic and electroneutral NBCs in the
villus cell type of the duodenal epithelium. Apparently, NBCs are not
expressed by the crypt cells of the murine duodenum. Further studies
are required to verify the proposed function and direction of transport
of the basolateral NBCs in a villus cell monolayer preparation or in
the intact epithelium.
|
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Ole Madsen, Mette Fredenslund, and Annette K. Rasmussen for skilled technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported by the following foundations: Lily B. Lunds Fond; Overlægerådet Odense Universitetshospital; Direktør Ib Henriksens Fond; Nygård Fonden, Katrine and Vigo Skovgaards Fond, Hørslev-Fonden, and Danish Medical Research Council (9601778).
Address for reprint requests and other correspondence: J. Praetorius, 10 Center Drive, Bldg. 10, Rm. 6N323, Bethesda, MD 20892 (E-mail: praetorj{at}nhlbi.nih.gov).
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 27 January 2000; accepted in final form 9 October 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aalkjaer, C,
and
Hughes A.
Chloride and bicarbonate transport in rat resistance arteries.
J Physiol (Lond)
436:
57-73,
1991[Abstract].
2.
Abuladze, N,
Lee I,
Newman D,
Hwang J,
Boorer K,
Pushkin A,
and
Kurtz I.
Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter.
J Biol Chem
273:
17689-17695,
1998
3.
Ainsworth, MA,
Amelsberg M,
Hogan DL,
and
Isenberg JI.
Acid-base transport in isolated rabbit duodenal villus and crypt cells.
Scand J Gastroenterol
31:
1069-1077,
1996[ISI][Medline].
4.
Ainsworth, MA,
Hogan DL,
Rapier RC,
Amelsberg M,
Dreilinger AD,
and
Isenberg JI.
Acid/base transporters in human duodenal enterocytes.
Scand J Gastroenterol
33:
1039-1046,
1998[ISI][Medline].
5.
Allen, A,
Flemström G,
Garner A,
and
Kivilaakso E.
Gastroduodenal mucosal protection.
Physiol Rev
73:
823-857,
1993
6.
Ameen, NA,
Ardito T,
Kashgarian M,
and
Marino CR.
A unique subset of rat and human intestinal villus cells express the cystic fibrosis transmembrane conductance regulator.
Gastroenterology
108:
1016-1023,
1995[ISI][Medline].
7.
Ameen, NA,
Martensson B,
Bourguinon L,
Marino C,
Isenberg J,
and
McLaughlin GE.
CFTR channel insertion to the apical surface in rat duodenal villus epithelial cells is upregulated by VIP in vivo.
J Cell Sci
112:
887-894,
1999
8.
Bevensee, MO,
Apkon M,
and
Boron WF.
Intracellular pH regulation in cultured astrocytes from rat hippocampus II Electrogenic Na/HCO3 cotransport.
J Gen Physiol
110:
467-483,
1997
9.
Boron, WF,
and
Boulpaep EL.
Intracellular pH regulation in the renal proximal tubule of the salamander basolateral HCO transport.
J Gen Physiol
81:
53-94,
1983[Abstract].
10.
Boyarsky, G,
Ganz MB,
Sterzel RB,
and
Boron WF.
pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO.
Am J Physiol Cell Physiol
255:
C844-C856,
1988
11.
Burnham, CE,
Amlal H,
Wang Z,
Shull GE,
and
Soleimani M.
Cloning and functional expression of a human kidney Na+:HCO cotransporter.
J Biol Chem
272:
19111-19114,
1997
12.
Cabantchik, ZI,
and
Greger R.
Chemical probes for anion transporters of mammalian cell membranes.
Am J Physiol Cell Physiol
262:
C803-C827,
1992
13.
Choi, I,
Aalkjaer C,
Boulpaep EL,
and
Boron WF.
An electroneutral sodium/bicarbonate cotransporter NBCn1 and associated sodium channel.
Nature
405:
571-575,
2000[ISI][Medline].
14.
Choi, I,
Romero MF,
Khandoudi N,
Bril A,
and
Boron WF.
Cloning and characterization of a human electrogenic Na+-HCO cotransporter isoform (hhNBC).
Am J Physiol Cell Physiol
276:
C576-C584,
1999
15.
Clarke, LL,
and
Harline MC.
Dual role of CFTR in cAMP-stimulated HCO secretion across murine duodenum.
Am J Physiol Gastrointest Liver Physiol
274:
G718-G726,
1998
16.
Eladari, D,
Blanchard A,
Leviel F,
Paillard M,
Stuart-Tilley AK,
Alper SL,
and
Podevin RA.
Functional and molecular characterization of luminal and basolateral Cl/HCO
exchangers of rat thick limbs.
Am J Physiol Renal Physiol
275:
F334-F342,
1998
17.
Hogan, DL,
Crombie DL,
Isenberg JI,
Svendsen P,
Schaffalitzky de Muckadell OB,
and
Ainsworth MA.
Acid-stimulated duodenal bicarbonate secretion involves a CFTR-mediated transport pathway in mice.
Gastroenterology
113:
533-541,
1997[ISI][Medline].
18.
Hoogerwerf, WA,
Tsao SC,
Devuyst O,
Levine SA,
Yun CHC,
Yip JW,
Cohen ME,
Wilson PD,
Lazenby AJ,
Tse CM,
and
Donowitz M.
NHE2 and NHE3 are human and rabbit intestinal brush-border proteins.
Am J Physiol Gastrointest Liver Physiol
270:
G29-G41,
1996
19.
Isenberg, JI,
Ljungström M,
Säfsten B,
and
Flemström G.
Proximal duodenal enterocyte transport: evidence for Na+-H+ and Cl-HCO
exchange and NaHCO3 cotransport.
Am J Physiol Gastrointest Liver Physiol
265:
G677-G685,
1993
20.
Ishiguro, H,
Steward MC,
Lindsay AR,
and
Case RM.
Accumulation of intracellular HCO by Na+-HCO
cotransport in interlobular ducts from guinea-pig pancreas.
J Physiol (Lond)
495:
169-178,
1996[Abstract].
21.
Ishiguro, H,
Steward MC,
Wilson RW,
and
Case RM.
Bicarbonate secretion in interlobular ducts from guinea-pig pancreas.
J Physiol (Lond)
495:
179-191,
1996[Abstract].
22.
Linsdell, P,
and
Hanrahan JW.
Disulphonic stilbene block of cystic fibrosis transmembrane conductance regulator Cl channels expressed in a mammalian cell line and its regulation by a critical pore residue.
J Physiol (Lond)
496:
687-693,
1996[Abstract].
23.
Marino, CR,
Jeanes V,
Boron WF,
and
Schmitt BM.
Expression and distribution of the Na+-HCO cotransporter in human pancreas.
Am J Physiol Gastrointest Liver Physiol
277:
G487-G494,
1999
24.
Maunsbach, AB,
Vorum H,
Kwon T-H,
Nielsen S,
Simonsen B,
Choi I,
Schmitt BM,
Boron WF,
and
Aalkjaer C.
Immunoelectron microscopical localization of the electrogenic Na,HCO3 cotransporter in rat and Ambystoma kidney.
J Am Soc Nephrol
11:
2179-2189,
2000
25.
Podolsky, DK,
and
Babyatsky MW.
Growth and development of the gastrointestinal tract.
In: Textbook of Gastroenterology, edited by Yamada T.. Philadelphia: JB Lippincott, 1995, p. 546-577.
26.
Praetorius, J,
Andreasen D,
Jensen BL,
Ainsworth MA,
Friis UG,
and
Johansen T.
NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells.
Am J Physiol Gastrointest Liver Physiol
278:
G197-G206,
2000
27.
Pushkin, A,
Abuladze N,
Lee I,
Newman D,
Hwang J,
and
Kurtz I.
Cloning, tissue distribution, genomic organization, and functional characterization of NBC3, a new member of the sodium bicarbonate cotransporter family.
J Biol Chem
274:
16569-16575,
1999
28.
Romero, MF,
and
Boron WF.
Electrogenic Na+/HCO cotransporters: cloning and physiology.
Annu Rev Physiol
61:
699-723,
1999[ISI][Medline].
29.
Romero, MF,
Fong P,
Berger UV,
Hediger MA,
and
Boron WF.
Cloning and functional expression of rNBC, an electrogenic Na+-HCO cotransporter from rat kidney.
Am J Physiol Renal Physiol
274:
F425-F432,
1998
30.
Romero, MF,
Hediger MA,
Boulpaep EL,
and
Boron WF.
Expression cloning and characterization of a renal electrogenic Na+/HCO cotransporter.
Nature
387:
409-413,
1997[ISI][Medline].
31.
Schmitt, BM,
Biemesderfer D,
Romero MF,
Boulpaep EL,
and
Boron WF.
Immunolocalization of the electrogenic Na+-HCO cotransporter in mammalian and amphibian kidney.
Am J Physiol Renal Physiol
276:
F27-F38,
1999
32.
Seidler, U,
Blumenstein I,
Kretz A,
Viellard-Baron D,
Rossmann H,
Colledge WH,
Evans M,
Ratcliff R,
and
Gregor M.
A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca2+-dependent HCO secretion.
J Physiol (Lond)
505:
411-423,
1997[Abstract].
33.
Seidler, U,
Hübner M,
Roithmaier S,
and
Classen M.
pHi and HCO dependence of proton extrusion and Cl
-base exchange in isolated rabbit parietal cells.
Am J Physiol Gastrointest Liver Physiol
266:
G759-G766,
1994
34.
Simson, JNL,
Merhav A,
and
Silen W.
Alkaline secretion by amphibian duodenum. II. Short-circuit current and Na+ and Cl fluxes.
Am J Physiol Gastrointest Liver Physiol
240:
G472-G479,
1981
35.
Simson, JNL,
Merhav A,
and
Silen W.
Alkaline secretion by amphibian duodenum. I. General characteristics.
Am J Physiol Gastrointest Liver Physiol
240:
G401-G408,
1981
36.
Sundaram, U.
Mechanism of intestinal absorption. Effect of clonidine on rabbit ileal villus and crypt cells.
J Clin Invest
95:
2187-2194,
1995[ISI][Medline].
37.
Thomas, JA,
Buchsbaum RN,
Zimniak A,
and
Racker E.
Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.
Biochemistry
18:
2210-2218,
1979[ISI][Medline].
38.
Tsumura, T,
Hazama A,
Miyoshi T,
Ueda S,
and
Okada Y.
Activation of cAMP-dependent Cl currents in guinea-pig paneth cells without relevant evidence for CFTR expression.
J Physiol (Lond)
512:
765-777,
1998
39.
Vorum, H,
Kwon T-H,
Fulton C,
Simonsen B,
Choi I,
Boron WF,
Maunsbach AB,
Nielsen S,
and
Aalkjaer C.
Immunolocalization of the electroneutral Na-HCO cotransporter (NBCn1) in medullary thick ascending limb and collecting duct intercalated cells in rat kidney.
Am J Physiol Renal Physiol
279:
F901-F909,
2000
40.
Yao, B,
Hogan DL,
Bukhave K,
Koss MA,
and
Isenberg JI.
Bicarbonate transport by rabbit duodenum in vitro: effect of vasoactive intestinal polypeptide, prostaglandin E2, and cyclic adenosine monophosphate.
Gastroenterology
104:
732-740,
1993[ISI][Medline].