School of Optometry, Indiana University, Bloomington, Indiana 47401
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ABSTRACT |
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Functional studies
support the presence of the Na+-HCO3
cotransporter (NBC) in corneal endothelium and possibly corneal
epithelium; however, molecular identification and membrane localization
have not been reported. To test whether NBC is expressed in bovine cornea, Western blotting was performed, which showed a single band at
~130 kDa for freshly isolated and cultured endothelial cells, but no
band for epithelium. Two isoforms of NBC have recently been cloned in
kidney (kNBC) and pancreas (pNBC). RT-PCR was run using cultured and
fresh bovine corneal endothelial and fresh corneal epithelial total RNA
and specific primers for kNBC and pNBC. RT-PCR analysis for pNBC was
positive in endothelium and weak in epithelium. The RT-PCR product was
subcloned and confirmed as pNBC by sequencing. No specific bands for
kNBC were obtained from corneal cells. Indirect immunofluorescence and
confocal microscopy indicated that NBC locates predominantly to the
basolateral membrane in corneal endothelial cells. Furthermore,
Na+-dependent HCO3
fluxes and
HCO3
-dependent cotransport with Na+ were
elicited only from the basolateral side of corneal endothelial cells.
Therefore, we conclude that pNBC is present in the basolateral membrane
of both fresh and cultured bovine corneal endothelium and weakly
expressed in the corneal epithelium.
sodium bicarbonate cotransporter; cornea; endothelium; bicarbonate transport
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INTRODUCTION |
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CORNEAL HYDRATION AND
TRANSPARENCY are maintained by the ion and fluid transport
properties of the corneal endothelium. Fluid transport is dependent on
the presence of HCO3 and significantly slowed by
carbonic anhydrase inhibitors (16, 19, 22, 28) and
stilbene anion transport inhibitors (24). Although it is
known that the secretion of HCO3
across endothelial
cells from basolateral (stroma) to apical (aqueous humor) cell
membranes gives rise to a small apical side negative transepithelial
potential (16, 19), the individual components of the
secretory process have not been fully described.
Functional Na+-HCO3 cotransport was first
identified in the salamander Ambystoma tigrinum
kidney (9) and has since been documented functionally in
numerous other cell types (31). In addition to coupled
fluxes of Na+ and HCO3
(37),
Na+-HCO3
cotransport is characterized by
being (31) 1) independent of Cl
,
2) inhibited by stilbenes, and 3) electrogenic;
however, an electroneutral version may be present in the heart
(13). More recently, the possibility of an electroneutral,
DIDS-insensitive version of the Na+-HCO3
cotransporter (NBC) has also emerged (27). In the kidney,
the stoichiometry of the electrogenic form of
Na+-HCO3
cotransport has been estimated
to be either 1Na+:2HCO3
or
1Na+:3HCO3
(35, 36).
However, recent oocyte expression studies of kidney NBC (kNBC)
indicated that the stoichiometry was
1Na+:2HCO3
(18, 34).
Previous studies (4, 5, 21) have shown that bovine corneal
endothelial cells (BCEC) take up HCO3 by a potent
Na+-dependent, DIDS-sensitive, electrogenic transporter.
This transport process is active in corneal endothelial intracellular
pH (pHi) regulation and presumably involved in
HCO3
secretion. That this transporter is
participating in net HCO3
uptake was indicated by
three findings: 1) steady-state pHi is higher in
the presence of HCO3
, 2) application of
basolateral DIDS always reduced pHi, and 3) steady-state intracellular Na+ concentration
([Na+]i) is significantly higher in the
presence of HCO3
(4). Given the
steady-state gradients of HCO3
and Na+,
together with the resting membrane potential of corneal endothelial cells, an NBC with a 1:2 stoichiometry, but not 1:3, would provide for
HCO3
influx (4).
Although the functional characteristics of
Na+-HCO3 cotransport have been described
in corneal endothelial cells, the molecular identification and membrane
localization have not been reported. Rat kidney (11, 32),
human kNBC [1,035 amino acids (aa), 116 kDa] (10), rat
pancreas (38), and human pancreas NBC (pNBC, 1,079 aa, 122 kDa) (1) have recently been cloned. The
NH2-terminal sequence of the two isoforms are different
(pNBC, MEDE; kNBC, MSTE), and pNBC contains additional
cAMP-dependent protein kinase A and protein kinase C sites. We used
known kNBC and pNBC sequences to study the expression of NBCs in bovine
corneal endothelium and epithelium. Furthermore, with the use of
NBC-specific antibodies, we confirmed functional assays that indicated
that a Na+-HCO3
uptake mechanism is
present on the basolateral membranes of fresh and cultured bovine
corneal endothelium.
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MATERIALS AND METHODS |
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Cell culture. BCEC were cultured to confluence onto 25-mm round coverslips, 13-mm Anodiscs, or T-25 flasks as previously described (3, 25). Briefly, primary cultures from fresh cow eyes were established in T-25 flasks with 3 ml of Dulbecco's modified Eagle's medium, 10% bovine calf serum, and antibiotic antimycotic (penicillin 100 U/ml, streptomycin 100 U/ml, and Fungizone 0.25 ug/ml), gassed with 5% CO2-95% air at 37°C and fed every 2 to 3 days. Primary cultures were subcultured to three T-25 flasks and grown to confluence in 5 to 7 days. The resulting second passage cultures were then further subcultured onto coverslips and allowed to reach confluence within 5 to 7 days.
Immunoblotting. Fresh BCEC and corneal epithelial cells were scraped from dissected cow corneas that had been kept on ice for 2-3 h since death. The cell scrapings were placed into ice-cold PBS that contained a protease inhibitor cocktail (Complete, Boehringer Mannheim) and were centrifuged at low speed for ~5 min. Cell pellets were resuspended in 2% SDS sample buffer that contained protease inhibitors. Cultured cells were dissolved directly in sample buffer. Both preparations were sonicated (Branson 250) briefly on ice and then centrifuged at 6,000 g for 5 to 10 min. An aliquot of the supernatant was taken for protein assay using the Lowry method (Bio-Rad). Five percent 2-mercaptoethanol and bromphenol blue were added to the remainder of the supernatant. The samples (not heated) were applied to an 8% polyacrylamide gel with a 4.5% stacking gel (60 ug/lane). After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane overnight at 4°C. Membranes were incubated in PBS that contained 5% nonfat dry milk for 1 h at room temperature. The blots were then incubated with rabbit polyclonal antibodies derived from maltose-binding protein (MBP)-NBC fusion peptides anti-MBP-NBC-3 (aa 338-391) or anti-MBP-NBC-5 (aa 928-1,035, the COOH terminus; a kind gift from B. M. Schmitt and W. F. Boron, 1:1,000) (33) in PBS that contained 5% nonfat dry milk for 1 h at room temperature with shaking. Next, the blots were washed five times for 5 min each with PBS/Tween 20, incubated with goat anti-rabbit secondary antibody coupled to horseradish peroxidase (Sigma) for 1 h at room temperature, washed with PBS/Tween 20 five times for 5 min each, and developed by enhanced chemiluminescence. Films were scanned to produce digital images that were then assembled and labeled using Microsoft PowerPoint software.
Immunofluorescence and confocal microscopy. Cultured cells grown to confluence on coverslips were washed three to four times with warmed (37°C) PBS and fixed for 30 min in warmed PLP fixation solution (2% paraformaldehyde, 75 mM lysine, 10 mM sodium periodate, and 45 mM sodium phosphate, pH 7.4) on a rocker. After fixation, the cells were washed three to four times with PBS. Coverslips were then kept for 5 min in PBS that contained 1% SDS to unmask epitopes and washed three times in PBS. Cells were blocked for 1 h in PBS that contained 0.2% bovine serum albumin, 5% goat serum, 0.01% saponin, and 50 mM NH4Cl. Rabbit polyclonal NBC antibody, diluted 1:100 in PBS/goat serum (1:1), was added onto coverslips and incubated for 1 h at room temperature. Preabsorption using MBP-NBC-3 and MBP-NBC-5 peptides (20 µg/ml) served as specific controls. Coverslips were washed three times for 15 min in PBS that contained 0.01% saponin. Secondary antibody conjugated to Oregon Green (Molecular Probes; 1:500) was applied for 1 h at room temperature. Coverslips were washed and mounted with Prolong antifade medium according to the manufacturer's (Molecular Probes) instructions.
To prepare fresh endothelial cells for immunofluorescence staining, cow corneas were dissected within ~2 to 3 h after death, washed with warmed PBS, and immediately fixed with warmed PLP fixation buffer for 10 min. Corneas were rinsed with PBS, and endothelium/Descemet's membrane strips were peeled off using jeweler's forceps and flattened onto Superfrost microscope slides (Fisher Scientific). Strips were fixed again at room temperature for 20 min and washed with PBS. The rest of the procedure was the same as for cultured cells. Fluorescence was observed with a standard epifluorescence microscope equipped with a cooled charge-coupled device camera. Fluorescence of selected specimens was documented with a Bio-Rad laser scanning confocal microscope to determine membrane localization.PCR primers. Oligonucleotides were obtained from GIBCO-Life Technologies. A pair of rat kNBC primers was constructed on the basis of the published cDNA sequence of rat kidney NBC from GenBank (accession no. AF027362). The kNBC sense primer (nucleotides 56-85 of mRNA) was 5'-ATGTCCACTGAAAATGTGGAAGGGAAGCCC-3' and the kNBC antisense primer (nucleotides 973-944 of mRNA) was 5'-GTCAGACATCAAGGTGGCGATGGCTCTTCC-3'. Another pair of human pNBC primers was also designed on the basis of the published cDNA sequence of human pancreas NBC from GenBank (accession no. AF069150). The pNBC sense primer (nucleotides 45-74) was 5'-ATGGAGGATGAAGCTGTCCTGGACAGAGGG-3' and the pNBC antisense primer (nucleotides 1,094-1,065) was 5'-ATCAGACATCAGGGTGGCAATGGCTCTGCC-3'. The expected lengths of RT-PCR products for kNBC and pNBC were, respectively, 918 and 1,050 bp.
RT-PCR. Total RNA was extracted from cultured and fresh bovine corneal endothelium using TRIzol reagent (GIBCO BRL) according to the manufacturer's instructions. Total RNA extracted from fresh bovine kidney cortex served as a positive control. To generate the first-strand cDNA, extracted total RNA (0.5-5 µg) was reverse-transcribed (total incubation mixture, 20 µl) at 42°C for 50 min in first-strand buffer (50 mM Tris, 75 mM KCl, and 3.0 mM MgCl2, pH 8.4) that contained 10 mM dithiothreitol, 0.5 mM of each dNTP, random primer (3 µg/ul, 1.5 µl; GIBCO BRL), and SuperScript II RT (40 U/µl, GIBCO BRL). First-strand cDNA was used in PCR amplification reactions (total incubation mixture, 25 µl) in a reaction buffer that contained 20 mM Tris (pH 8.4), 50 mM KCl, 2.0 mM MgCl2, TaKaRa Ex Taq polymerase (2.5 units, TaKaRa Shuzo), and 0.2 mM of each dNTP, with the specific primer set described above. Final concentration of primers for kNBC and pNBC was 0.1 µM. Nanopure water substituting for first-strand cDNA served as negative control.
PCR amplifications were carried out in a thermocycler under the following conditions: denaturation at 94°C for 3 min for one cycle, 30 cycles of denaturation at 94°C for 1 min each, annealing at 61°C for 1 min, extension at 72°C for 2 min, and a final extension for one cycle at 72°C for 15 min. The PCR products were loaded onto 1% agarose gel, electrophoresed, and stained with 0.5 µg/ml ethidium bromide.Subcloning and sequencing. Approximately 1 kb pNBC PCR product was purified using a 1% low-melting point agarose gel. Freshly purified products were mixed for 5 min with pCR 4-TOPO vector (Invitrogen, San Diego, CA). The TOPO cloning reaction was added into a vial of One Shot cells for plasmid transformation. The transformed bacteria were plated on agar culture media that contained ampicillin (50 ug/ml) and incubated at 37°C overnight. Inserts from selected clones were digested with EcoR I (GIBCO BRL), and size was confirmed using 1% agarose minigel electrophoresis. The vectors with predicted inserts were isolated using a plasmid miniprep kit (Qiagen, Chatsworth, CA). Sequencing was performed using the ABI Prism BigDye terminator cycle sequencing ready reaction mix (PE Applied Biosystems) according to the manufacturer's instructions. Sequencing electrophoresis was run on the ABI Prism 377 DNA sequencer in the Indiana University Molecular Biology Institute. Sequences were assembled and compared using Vector NTI version 5.2 software (InforMax, North Bethesda, MD).
Microscope perfusion.
For measurement of pHi and [Na+]i
using fluorescent dyes (see Measurement of pHi
and Measurement of
[Na+]i),
cells cultured to confluence on AnoDisc membranes were placed in a
double-sided perfusion chamber designed for independent perfusion of
the apical and basolateral sides (8). The assembled
chamber was placed on a water-jacketed (37°C) brass collar held on
the stage of an inverted microscope (Nikon Diaphot) and viewed with a
long working distance (1.2 mm) water-immersion objective (Zeiss, ×40).
Apical and basolateral compartments were connected to hanging syringes
that contained Ringer solution in a Plexiglas warming box (37°C)
using Phar-Med tubing. The flow of the perfusate (~0.5 ml/min) was
achieved by gravity. Two independent eight-way valves were employed to
select the desired perfusate for the apical and basolateral chambers.
The composition of the HCO3-rich Ringer solution used
throughout the study was (in mM) 150 Na+, 4 K+,
0.6 Mg2+, 1.4 Ca2+, 118 Cl
, 1 HPO4
, 10 HEPES
, 28.5 HCO3
, 2 gluconate
, and 5 glucose,
equilibrated with 5% CO2 and pH adjusted to 7.50 at
37°C. HCO3
-free Ringer solution (pH 7.50) was
prepared by equimolar substitution of NaHCO3 with
sodium gluconate. Na+-free HCO3
-rich
Ringer solution was prepared by substitution of NaCl with 50 mM KCl,
balance N-methyl-D-glucamine chloride, and
substitution of NaHCO3 with KHCO3.
Osmolarity was adjusted to 300 ± 5 mosM with sucrose.
Measurement of pHi.
BCEC cultured onto permeable Anodisc filters were loaded with the
pH-sensitive fluorescent dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)
by incubation in HCO3-free Ringer solution that
contained 1-5 µM BCECF-AM at room temperature for 30-60
min. Dye-loaded cells were then kept in Ringer solution for at least 30 min before use. Fluorescence excitation (495 and 440 nM) and data
collection were obtained using a DeltaRam ratio fluorescence
system (Photon Technology, Monmouth Junction, NJ) controlled by Felix
software. Fluorescence ratios were obtained at 1 s
1 and
were calibrated against pHi by the high potassium-nigericin technique (39). A calibration curve, which follows a pH
titration equation, has been constructed for BCEC (3).
Measurement of [Na+]i. [Na+]i was measured with the Na+-sensitive fluorescent dye sodium-binding benzofuran isophthalate (SBFI; Molecular Probes, Eugene, OR). The cells cultured onto Anodiscs were loaded by incubation in Ringer solution that contained 5-10 µM SBFI-AM and 0.01% Pluronic F-127 at room temperature for ~1 h. Dye-loaded cells were then kept in Ringer solution for at least 30 min before use. The dye was alternately excited at 350 and 385 nm with a 13-nm bandpass. The 350-nm wavelength, rather than the typical 340-nm wavelength, was used to enhance transmission of the excitation light through the glass optics of the long working distance objective. The dichroic mirror was centered at 400 nm, and the barrier filter was a 420- to 500-nm bandpass. Calibration of the fluorescence ratio against [Na+]i was performed as previously described (4, 26).
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RESULTS |
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To confirm the presence of the NBC in corneal endothelium, we used
immunoblotting with rabbit anti-NBC antibody. Figure
1 shows that the MBP-NBC-3
antibody gave positive bands for cultured and fresh corneal endothelium
at ~130 kDa, which is the expected range for mammalian NBC
(33). The MBP-NBC-5 antibody gave the same result (not
shown). As demonstrated previously with kidney (33),
preabsorption with the respective fusion proteins eliminated the bands
(not shown). There has been one report of possible NaHCO3 cotransport activity in corneal epithelium (12). However,
in our study, the immunoblots were routinely negative. Figure 1 also indicates that the apparent density of NBC bands was considerably higher for the fresh cell preparations than for the cultured cells.
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Figure 2A shows
immunofluorescence distribution of NBC in cultured and fresh bovine
corneal endothelium. MBP-NBC-3 polyclonal antibody (as well as
MBP-NBC-5, not shown) revealed an apparent basolateral membrane
location in fresh and cultured bovine corneal endothelium. Figure
2B shows that immunofluorescence was significantly inhibited
by fusion protein preabsorption. Basolateral localization was confirmed
by confocal microscopy. Figure 3 shows
sequential confocal images from basal to apical membranes (total cell
thickness ~4 µm). Strong NBC immunostaining is seen toward the
basal and lateral membranes in fresh bovine corneal endothelium, with
no apparent apical staining. These results are consistent with previous functional studies [e.g., basolateral application of DIDS causing cell
acidification (8)] that indicated that
Na+:HCO3 cotransport should be on the
basolateral membrane.
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Further functional evidence for a basolateral NBC is shown in
Figs. 4 and
5. Figure 4A shows the effect
on pHi after removal of
CO2-HCO3 sequentially from the apical
side and then the basolateral side. On both the apical and basolateral
sides, there was an initial rapid alkalinization due to rapid efflux of
CO2. On the apical side, the alkalinization was larger and
was followed by a small acidification. A new steady-state
pHi was reached within 2 to 3 min and was always above the
baseline pHi, as was shown previously (5).
When CO2-HCO3
-rich Ringer solution was
reintroduced, there was a rapid acidification, small undershoot, and
then return to the baseline pHi. On the basolateral side,
the alkalinization was small and followed by a significant
acidification and then recovery to a new steady-state pHi
below the baseline. When CO2-HCO3
-rich
Ringer solution was reintroduced, there was a small acidification and
robust alkalinization over the baseline that returned within a few
minutes (not shown). The smaller initial alkalinization and deeper and
more rapid acidification on basolateral
CO2-HCO3
removal, and similarly, the much
greater alkalinization on basolateral CO2-HCO3
reintroduction, indicate that
HCO3 permeability is greater on the basolateral side than
on the apical side (5). To test whether the
HCO3
fluxes are Na+ dependent, we
repeated the experiment using cells perfused with a high
K+, Na+-free Ringer solution. Figure
4B shows that in the absence of Na+, the apical
response is similar to that of the control (Fig. 4A). On the
basolateral side, there was the initial rapid alkalinization; however,
the sharp acidification below the baseline and the robust alkalinization on CO2-HCO3
reintroduction
did not occur. Similar results were obtained in six other experiments.
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Figure 5 shows the effects of HCO3 removal on
[Na+]i. When HCO3
was
removed from the basolateral side, [Na+]i
decreased from 30 to ~17 mM (mean
[Na+]i =
14 ± 5, n = 5) and increased to ~32 mM when
CO2-HCO3
-rich Ringer solution was
reintroduced. On the apical side, there was an increase of
[Na+]i from 32 to 47 mM when
HCO3
was removed (mean
[Na+]i = +11 ± 6, n = 5). This result shows that
HCO3
-dependent cotransport with Na+ is
occurring only on the basolateral side.
Because different isoforms of NBC have been cloned in various tissues,
we asked which of the two major isoforms (kNBC or pNBC) is expressed in
bovine corneal endothelium. RT-PCR was performed using specific primers
for kNBC and pNBC with the use of fresh and cultured bovine corneal
endothelium RNA. Figure 6A
shows that RT-PCR gave positive bands at ~1 kb for pNBC from fresh
and cultured bovine corneal endothelium. On the other hand, corneal
epithelium gave a very weak positive band. Figure 6B shows
that RT-PCR using primers for kNBC gave negative results for fresh and
cultured bovine corneal endothelium and fresh bovine corneal
epithelium. Figure 6B also shows that fresh bovine kidney
cortex gave a strong positive band for kNBC and a weaker band for pNBC.
These results indicate that the kNBC primers, which were designed from
the rat kNBC sequence, were appropriate to probe for bovine kNBC.
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Sequencing confirmed the identity of pNBC in BCEC. The first 1,050 base pairs of pNBC published for other species were used for
alignment with our result. Nucleotide sequence alignment showed 93%
homology with human pancreas NBC (hpNBC). Figure
7 shows the amino acid alignment of our
product sequence with hpNBC, rat pancreas NBC, and rat kidney NBC.
Amino acid homology was 99% with hpNBC for the first 350 amino acids.
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DISCUSSION |
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The recent cloning of NBCs in other tissues has made it possible
to examine the expression of these transporters at the molecular level
in BCEC. Previous functional work has shown that
HCO3-dependent transport is essential for the
maintenance of corneal hydration (16, 19, 22, 28) and
regulation of pHi (4, 5, 21). Among several
transporters, such as the Na+/H+ exchanger and
Cl
/HCO3
exchanger, the
Na+-HCO3
cotransporter has been suggested
to play a key role in corneal endothelium HCO3
transport (5). In contrast to membrane vesicle studies
that were unable to show NBC in BCEC (23, 30), we clearly
demonstrated by immunoblot analysis, indirect immunofluorescence,
molecular identification, and functional assays that NBC is present and active in these cells.
Immunoblot analysis showed strong expression of NBC in fresh and
cultured corneal endothelium. On the other hand, fresh bovine corneal
epithelial tissue always gave a negative immunoblot result. Interestingly, the density of the band from cultured endothelium was
consistently weaker than fresh endothelium, which suggests that
Na+-HCO3 cotransport is more active in
fresh bovine corneal endothelium. Further functional assays of NBC in
fresh tissue are needed to confirm this possibility.
The antibody used in this study cannot distinguish between kNBC
and pNBC; however, the RT-PCR results and sequencing indicate strong
expression of pNBC for fresh and cultured bovine corneal endothelium. The weak expression of pNBC in fresh epithelial cells may
indicate that membrane protein purification could yield a positive
immunoblot identification. Nevertheless, this result shows that
significant Na+-HCO3 cotransport is not
likely in the corneal epithelium. RT-PCR for kNBC was always negative
for corneal endothelium and epithelium, which is consistent with kNBC
being specifically expressed in kidney, whereas pNBC expression is more
widespread (31). Our results contrast with a recent report
that shows positive RT-PCR results for both pNBC and kNBC in human
corneal endothelium (40); however, these products were not
confirmed by sequencing. Preliminary studies from our laboratory have
failed to find kNBC expression in human corneal endothelium.
Localization of NBC by indirect immunofluorescence and confocal
microscopy showed a basolateral distribution in both fresh and cultured
BCEC. Inspection of the basal staining shows an uneven granular
appearance that may represent intracellular rather than basal membrane
NBC. On the other hand, the apical side shows no apparent NBC
expression. Furthermore, Na+-dependent
HCO3 fluxes could be demonstrated on the basolateral
side but not on the apical side (see Fig. 4). Likewise, Na+
fluxes cotransported with HCO3
were readily observed
across the basolateral membrane (see Fig. 5). On the other hand,
HCO3
efflux across the apical membrane caused an
increase in [Na+]i. This is unlikely to be
due to Na+/H+ exchange since this exchanger is
essentially inactive at the resting pHi of endothelial
cells (3) and also because apical HCO3
removal causes a net increase in pHi, which would further
inhibit Na+/H+ activity (2). The
simplest interpretation is that HCO3
removal at the
apical membrane causes a transient drop in intracellular HCO3
concentration or membrane
depolarization (6), thereby increasing the driving force
for NaHCO3 entry across the basolateral membrane. Taken
together, these results show conclusively that
Na+-HCO3
cotransport is present and
active in the basolateral membranes of BCEC.
The model for transendothelial HCO3 transport remains
incomplete. Figure 8 shows that a number
of transporters have been identified on the basolateral membrane, but
little is known about apical transport. In addition to
Na+-HCO3
cotransport, the
Na+-K+-ATPase (41), the
Na+:K+:2Cl
cotransporter
(14, 20), the Na+/H+ exchanger
(3, 30), and various organic anion transporters (15,
17) have been localized to the basolateral membrane. Although it
is conceivable that Na+/H+ exchange could
contribute to basolateral HCO3
uptake, it is unlikely
since the high pHi of endothelial cells inhibits
Na+/H+ exchange activity (3).
Furthermore, the Na+/H+ exchange blocker
amiloride has no effect on resting endothelial pHi in the
presence of HCO3
(4), and 0.5 mM
amiloride had no apparent short-term effect on endothelial fluid
transport (42). Thus NBC is the predominate mechanism for
basolateral HCO3
uptake. Contribution to apical
HCO3
efflux may include the anion exchanger
(8), conductive flux through anion channels
(6), and/or carbonic anhydrase-mediated conversion of
CO2 efflux to HCO3
(5). What
is clear, however, is that apical HCO3
permeability
is significantly lower than basolateral permeability (5).
Thus the rate-limiting step in transendothelial HCO3
transport is at the apical membrane, which will need to be the focus of
future investigation.
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ACKNOWLEDGEMENTS |
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We thank B. M. Schmitt and W. F. Boron, Dept. of Cellular and Molecular Physiology, School of Medicine, Yale Univ., for kindly providing the antibodies used in this study. We also thank M. Romero, Dept. of Physiology and Biophysics, Case Western Univ., for helpful comments on PCR and subcloning procedures.
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FOOTNOTES |
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This work was supported by National Eye Institute Grant EY-08834 (to J. A. Bonanno).
Address for reprint requests and other correspondence: J. A. Bonanno, School of Optometry, Indiana Univ., 800 E. Atwater Ave., Bloomington, IN 47401 (E-mail: jbonanno{at}indiana.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 13 March 2000; accepted in final form 28 June 2000.
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