Expression and localization of Na+-HCO3minus cotransporter in bovine corneal endothelium

Xing Cai Sun, Joseph A. Bonanno, Sergey Jelamskii, and Qiang Xie

School of Optometry, Indiana University, Bloomington, Indiana 47401


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Immunoblot of cultured (Cult Endo) and fresh bovine corneal endothelium (Fresh Endo) and fresh corneal epithelium (Fresh Epi) whole cell extracts using the maltose-binding protein (MBP)-Na+-HCO3- cotransporter (NBC)-3 antibody. Each lane contains 60 µg of protein.

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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Fig. 2.   Immunofluorescence localization of NBC. A: cultured bovine corneal endothelium (top) and fresh bovine corneal endothelium (bottom). B: immunofluorescence of cultured corneal endothelium using MBP-NBC-3 antibody preabsorbed with MBP-NBC-3 fusion protein (top) and MBP-NBC-3 antibody preabsorbed with MBP-NBC-5 fusion protein (bottom). These images were acquired with the same exposure time and printed using the same contrast and brightness settings. Scale bars are 35 µm.



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Fig. 3.   Laser scanning confocal immunofluorescence microscopy of fresh bovine corneal endothelium. A: the most basal image. Images B-F are successively 0.5 µm more apical than the image in A. Images beyond F showed no detectable fluorescence.

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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Fig. 4.   Na+ dependence of intracellular pH (pHi) change due to apical and basolateral CO2-HCO3- removal. A: control responses. Both apical and basolateral compartments were perfused with CO2-HCO3- Ringer solution. Boxes indicate when CO2-HCO3- Ringer solution was removed from the apical or basolateral side. B: same experiment as A, but in the absence of Na+.



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Fig. 5.   HCO3--dependent changes in intracellular Na+ concentration ([Na+]i) following apical or basolateral CO2-HCO3- removal. [Na+]i was measured using the fluorescent dye sodium-binding benzofuran isophthalate (see MATERIALS AND METHODS). Both apical and basolateral compartments were perfused with CO2-HCO3- Ringer solution. Boxes indicate when CO2-HCO3- Ringer solution was removed from the apical or basolateral side.

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 Delta [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 Delta [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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Fig. 6.   RT-PCR for pancreas NBC (pNBC) and kidney NBC (kNBC). A: probing for pNBC using fresh and cultured bovine corneal endothelium and fresh corneal epithelium show single bands at the expected size of ~1 kb. B: probing for kNBC using fresh and cultured bovine corneal endothelium and fresh corneal epithelium were negative (4th, 5th, and 6th lane from the left). Bovine kidney cortex (2nd lane from the left) was positive for kNBC at ~900 bp, indicating that the kNBC PCR primers could detect bovine kNBC. Detection of pNBC in kidney (3rd lane from the left) is consistent with its widespread distribution. Negative controls are shown at the far right (Neg Ctrl kNBC and Neg Ctrl pNBC).

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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Fig. 7.   Amino acid sequence (bcNBC) derived from nucleotide sequence of subcloned 1,050-bp RT-PCR fragment from bovine corneal endothelium. Sequence comparison with human pancreas (hpNBC) indicates ~99% homology for the first 350 amino acids. rkNBC is rat kidney and rpNBC is rat pancreas. Underlined amino acids are primer sequences used for RT-PCR. Solid background indicates complete homology. Italics indicate amino acid difference with hpNBC (amino acid 256). Shaded italics indicate no homology with the other NBCs (amino acids 52, 70, and 297).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 8.   Model for transendothelial HCO3- transport. Basolateral NBC loads endothelial cells with HCO3- from the stromal side. The carbonic anhydrase inhibitor acetazolamide acidifies endothelial cells indicating that there is normally net conversion of HCO3- to CO2 (7). Three possible modes for apical HCO3- efflux from cell to aqueous humor are shown: 1) Cl-/HCO3- exchange, 2) CO2 efflux and conversion to HCO3- by a membrane-bound carbonic anhydrase isozyme IV (CAIV), and/or 3) conductive flux via anion channels. Although Na+:K+:2Cl- cotransport is involved in endothelial cell volume regulation (14), its role in net fluid secretion appears to be small (29). CAII, carbonic anhydrase isozyme II.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   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[Abstract/Free Full Text].

2.   Aronson, P. Kinetic properties of the plasma membrane Na-H exchanger. Annu Rev Physiol 47: 545-560, 1985[ISI][Medline].

3.   Bonanno, JA, and Giasson C. Intracellular pH regulation in fresh and cultured bovine corneal endothelium. I. Na/H exchange in the absence and presence of HCO3-. Invest Ophthalmol Vis Sci 33: 3058-3067, 1992[Abstract].

4.   Bonanno, JA, and Giasson C. Intracellular pH regulation in fresh and cultured bovine corneal endothelium. II. Na:HCO3 cotransport and Cl/HCO3 exchange. Invest Ophthalmol Vis Sci 33: 3068-3079, 1992[Abstract].

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