2Ophthalmology and 3Physiology and Cellular Biophysics, Columbia University, New York, New York 10032; and 1Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07103
Submitted 2 December 2003 ; accepted in final form 6 February 2004
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ABSTRACT |
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confocal microscopy; cryosections; stoichiometry; pH titration; chloride channels
We recently reexamined the question of apical exit of HCO3 and, from theoretical considerations (unpublished observations), determined that for proper balance of electrical and chemical fluxes across these cells, coupled Na+-HCO3 apical exit [as postulated by Jentsch et al. (20)] could not be discarded. Moreover, we report here that inhibition of Cl channels as well as selective inhibition of the membrane-bound CA IV with impermeant CA inhibitors result in only small changes in corneal endothelial fluid transport and thus do not appear to be the major driving forces for it. In the search for other mechanisms for fluid transport, we therefore set out to examine the distribution of NBC proteins in cultured and fresh corneal endothelial cells. Here we present evidence that both basolateral and apical membranes express an NBC protein, and we discuss how different stoichiometries (2:1 entry, 3:1 exit) might underlie transcellular HCO3 flux.
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MATERIALS AND METHODS |
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Corneal tissue preparation.
Bovine eyes were obtained from an abattoir as previously described (9). Before dissection, the ocular globes were washed with PBS. The globes were placed in a petri dish, and the corneas were dissected off. Corneas were then washed twice with a HEPES-buffered Hanks' balanced salt solution (medium 199; catalog no. 12350, GIBCO-BRL) and were cut in rectangular pieces of 1 x 0.5 cm. These pieces were then washed three times with medium 199 at 37°C and fixed as described in Immunocytochemistry.
Immunocytochemistry. Cultured bovine corneal endothelial cells (CBCEC) subcultured as described in Cell culture were washed twice with medium 199 at 37°C, fixed for 30 min in PLP fixative (2% formaldehyde, 75 mM lysine, 10 mM sodium periodate, 45 mM sodium phosphate, pH 7.4; Ref. 28), washed in Dulbecco's PBS (GIBCO-BRL) three times, permeabilized with 0.075% saponin in PBS for 20 min, and washed again in PBS three times. The cells were then incubated for 1 min in a 1% solution of sodium dodecyl sulfate (SDS) in PBS. The cell monolayers were then incubated in blocking solution (15% goat serum, 0.3% Triton X-100, 20 mM sodium phosphate, 0.9 mM sodium chloride; Ref. 25) for 30 min. After the blocking solution was removed by aspiration, the monolayers were exposed to a rabbit anti-NBC polyclonal antibody (catalog no. AB3204, Chemicon International, Temecula, CA), which recognizes the rat kidney NBC-1 COOH terminus amino acids 9901035 segment, or to another rabbit anti-NBC polyclonal antibody (also from Chemicon, catalog no. AB3212), which recognizes the rat kidney NBC-1 NH2 terminus amino acids 338391 segment. The antibodies were diluted in PBS plus 15% goat serum and 0.2% bovine serum albumin (Sigma, St. Louis, MO) in PBS. After trying a range of concentrations, we settled on a dilution of 1:500 for the primary antibody (final concentration 2 µg/ml). As a control, in given experiments, we preabsorbed with the immunogenic NBC COOH-terminal peptide (amino acids 9901035; synthesized for us by Alpha Diagnostics International, San Antonio, TX). The final concentration of the peptide was 2 µg/ml (in PBS). The incubations were done for 60 min in a humid chamber at room temperature. Subsequently, the cell layers were washed three times in PBS and twice in PBS plus 5% goat serum (510 min each time). The cell layers were then incubated in rhodamine red-X-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:200 (in PBS) for 90 min in the same humid chamber at room temperature. Samples were prepared and kept in the dark to prevent light-induced damage to rhodamine red-X.
Tissue preparation. Corneal tissue samples were fixed in PLP medium for 1 h and were washed in PBS three times. They were then immersed in 30% sucrose and kept overnight at 4°C. Subsequently the excess solution was absorbed away, and the pieces were embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Sakura Finetek, Torrance, CA). The pieces were kept in a freezer at 20°C and were subsequently sectioned in a cryostat (CM 1850, Leica Microsystems, Bannockburn, IL; 4-µm-thick specimens). Specimens were collected on Silane-Prep slides (Sigma). The rest of the procedure was the same as described for the cultured cells, including permeabilization, denaturation, and blocking. For control experiments once more we used the antigen peptide.
Nuclear counterstaining and mounting. Samples were equilibrated briefly in 2x SSC solution (300 mM sodium chloride, 30 mM sodium citrate, pH 7.0 titrated with HCl) and then incubated for 20 min in 2x SSC solution containing 5 µg/ml DNase-free RNase (Boehringer Mannheim, Indianapolis, IN). Incubation was terminated with three washes in 2x SSC solution (1 min each), after which samples were incubated for 5 min in Sytox green (Molecular Probes, Eugene, OR) diluted 1:300 (vol/vol) in 2x SSC solution and then rinsed five times (1 min each) in 2x SSC solution. The upper part of the chamber slide system was then quickly removed, leaving only the cells on the slides. Finally, coverslips were applied to the samples with 1 drop per well of H-1000 Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA). The excess mounting medium was aspirated, and the coverslips were secured with clear nail polish.
The preparations were screened for fluorescence with an Axiovert S100 microscope (Carl Zeiss, Thornwood, NY) using excitation wavelengths of 480 and 545 nm to detect emission by nuclear staining (Sytox green) or antibody staining (rhodamine red-X), respectively. Preparations showing staining were then examined with a scanning confocal microscope (LSM 410, Carl Zeiss). Excitation came from its argon-krypton laser, producing lines at 488 or 568 nm. Fluorescence in the x-y plane was recorded at different depths, and fluorescence in the x-z and y-z planes was obtained from the combined x-y images with Zeiss LSM-PC software. The images were enhanced with Adobe Photoshop software (San Jose, CA).
Transendothelial HCO3 flux measurements. The HCO3 flux across cultured bovine endothelial cell monolayers was measured with a pH-stat method adapted and modified from van Adelsberg et al. (42). Permeable tissue culture inserts with confluent monolayers were mounted in a modified Ussing chamber (Fig. 1) consisting of two compartments separated by the insert membrane and the cell monolayer. Each compartment was connected to a reservoir, and solutions from the reservoirs circulated continuously through the compartments with the aid of an airlift system so that the chamber volume was exchanged once every 15 s. One compartment of the Ussing chamber was perfused with a HCO3-CO2-buffered solution aerated with a mixture of 5% CO2 and 95% air, whereas the other compartment was perfused with a solution nominally free of HCO3 and CO2, which was vigorously bubbled with CO2-free air and buffered with a low-concentration (0.1 mM) phosphate buffer. Except where noted, the detailed composition of the HCO3-CO2-buffered solution (solution II) was (in mM) 126.5 NaCl, 4.8 KCl, 1.7 CaCl2, 1.0 MgSO4, 1.0 NaH2PO4, 26.2 NaHCO3, and 5.5 glucose; pH was 7.4. The HCO3-CO2-free solution (solution I) contained (in mM) 154 NaCl, 4.8 KCl, 1.7 CaCl2, 1.0 MgSO4, 0.066 Na2HPO4, 0.033 NaH2PO4, and 5.5 glucose (pH 7.4). With this arrangement, CO2 and HCO3 diffused from solution II to solution I in parallel with any active transendothelial transport. In the phosphate-buffered compartment (solution I), the CO2 was driven off by the CO2-free air and the pH change resulting from the accumulating HCO3 was detected by a pH electrode and a pH meter. Because the pH of the compartment became more alkaline than the control pH (7.4, unless specified), the voltage output from the pH meter differed from a set level, triggering a stepping motor that in turn drove a microsyringe injecting 1 M HCl into the solution I compartment until its pH returned to the control value. Each step of the motor injected 1 nmol of HCl into solution I; the amount of steps accumulated was displayed on a LED display reset every 30 min. This automatic titration system maintained the pH of solution I constant within 0.08 pH units.
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A possible error of this method is due to the reversible reaction of CO2 diffusing from solution II to solution I to yield carbonic acid followed by subsequent dissociation of carbonic acid to H+ and HCO3. This error was kept low by bubbling the solution I vigorously with CO2-free air so as to maintain PCO2 as close to zero as possible. Moreover, because the hydration reaction is much slower than the dehydration reaction, the probability that some HCO3 is converted to CO2 and removed in the CO2-free atmosphere is higher than for the reverse reaction. This would then lead to an underestimate of the unidirectional HCO3 fluxes. However, because the transendothelial HCO3 transport is calculated as the difference of the unidirectional fluxes in the basolateral-to-apical and apical-to-basolateral directions, the errors would tend to cancel out.
Transendothelial fluid transport measurements. The putative role of the Cl channels and CO2 diffusion in the generation of fluid transport was investigated by the Maurice-Dikstein method (10), a method that determines fluid transport by monitoring corneal thickness. Corneal thickness is a function of the imbibition pressure generated by the stromal glycosaminoglycans, which drives fluid into the stroma, and by the fluid secretion mechanism of the corneal endothelium. In the physiologically stable cornea, these two mechanisms are equal so that there is no net fluid transport and no change in corneal thickness. However, any change in endothelial ion and fluid transport will be reflected in a change of corneal thickness.
Rabbit corneas were obtained from New Zealand albino rabbits (2 kg). Animal procedures were in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, revised 1985). Rabbits were euthanized with an overdose of pentobarbital sodium solution (Butler, Columbus, OH) injected into the marginal ear vein. The eyes were enucleated immediately by the technique of Dikstein and Maurice (10). The deepithelialized cornea was mounted in a Dikstein-Maurice chamber held in a thermal jacket kept at 36.5°C and viewed with a specular reflection microscope (10). The endothelial side of the cornea was superfused with solutions at a rate of 1.4 ml/h by a syringe pump (Razel Scientific Instruments, Stamford, CT), whereas the stromal surface was covered with silicone oil. Stromal thickness was measured in the conventional fashion (2) by focusing in sequence on the stroma-oil and endothelium-fluid interfaces and using the microscope micrometer to gauge their separation. Readings were taken every 15 min for 4 h.
For endothelial superfusion, the medium used was a HEPES-HCO3 Ringer solution containing (in mM) 104.4 NaCl, 26.2 NaHCO3, 3.8 KCl, 1 KH2PO4, 0.8 MgSO4, 1.7 CaCl2, 6.9 glucose, and 20 HEPES. The pH was 7.4, and the osmolality was 290 mosmol/kgH2O. Ouabain, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and parabromophenacyl bromide (pBPB) were from Sigma. The specific CFTR inhibitor (26) was a gift of Dr. Alan Verkman (University of California, San Francisco, CA). Benzolamide was kindly supplied by Dr. Eric Swenson (University of Washington, Seattle, WA); we also used dextran-bound acetazolamide (Synthelec, Lund, Sweden). CA was from bovine erythrocytes (catalog no. 215755, Calbiochem, La Jolla, CA). Stock solutions of the reagents were obtained by dissolving them in dimethyl sulfoxide (DMSO) (except for ouabain, which was dissolved in Ringer solution). The final concentration of DMSO in the superfusion medium was kept at 0.1%.
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RESULTS |
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In a typical experiment, HCO3 flux from the apical to the basolateral side (presumed direction of the leak) was measured for a period of 30 min. The solutions were then replaced on both sides of the preparation so as to determine HCO3 flux in the opposite direction (from basolateral to apical, with the expectation of determining active transport plus leak) again for 30 min. This was then followed by yet another solution exchange and a determination of apical-to-basolateral flux for 30 min. Thus there were two measurements of the presumed leak bracketing the measurement of active transport plus leak. We took the average of the two leak measurements and subtracted it from the basolateral-to-apical flux, which gave us the putative transcellular net HCO3 flux for that experiment. Figure 2 shows the values for the unidirectional fluxes (basolateral to apical and apical to basolateral) and the net fluxes. The HCO3 flux from the basolateral to the apical side was consistently greater than the reverse flux, resulting in a statistically significant net flux of 0.53 ± 0.07 µeq·h1·cm2 (Fig. 2). In addition, from the passive unidirectional flux values for the cell monolayer on its insert and the insert alone and the HCO3 concentration ([HCO3]) gradient, we calculated the permeability of the cell monolayer to HCO3 to be 4.8 (±0.2) x 105 cm/s.
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Localization of NBC: cultured cells. We investigated the localization of NBC both in cultured cells and in fresh corneal tissue slices. Figure 6 depicts selected images of a gallery of optical sections of CBCEC obtained by confocal microscopy. Evidence for the presence of NBC is seen at the level of the apical membrane (Fig. 6, a and b), lateral membrane (Fig. 6c), and lateral and basal membrane (Fig. 6d). The three-dimensional reconstruction shown in Fig. 7 reinforces this view. Figure 7b, the xz reconstruction, is especially noteworthy in that the stained membrane envelope all around a cell is clearly noticeable. There is also some diffuse staining of the cytoplasm. This gallery is representative of 11 galleries obtained in cells from two different batches of bovine eyeballs.
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DISCUSSION |
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These observations justify the conclusion that the cultured cells are polarized and exhibit net HCO3 transport that would correspond to a distribution and a density of expression of HCO3 transporters similar or identical to those of fresh tissue. This net HCO3 transport would then form part of the fluid transport machinery in this layer; a recent model (33) includes a role for electrogenic transcellular HCO3 transport in electroosmosis-driven transendothelial fluid transport.
Inhibition of Cl channels. Several types of anion channels have been described for corneal endothelial cells. These channels include a large-conductance nonspecific anion channel identified by patch-clamp studies (30), which is inoperative at resting membrane potential. In addition, RT-PCR screening has revealed the presence and activation of CFTR (38) and CaCC (43) channels in the bovine corneal endothelium.
Inhibition of Cl channels with a range of different Cl channel inhibitors (Fig. 4) consistently showed only relatively small effects on transendothelial fluid transport, ranging from insignificant to a maximum of 16%. The contribution of apical Cl channels to transendothelial fluid transport is of the same order of magnitude as that of the basolateral Na+-K+-2Cl cotransporter (NKCC) (21), suggesting the existence of a small vectorial Cl transport from the basolateral to the apical side.
Inhibition of CA IV.
The corneal endothelium possesses a membrane-bound extracellular CA IV (31), which is confined to the apical membrane (6). It has been hypothesized that this CA IV catalyzes the hydration of CO2 to HCO3 in the unstirred layer adjacent to the apical membrane and thus creates a stromal-to-aqueous CO2 gradient; it has been postulated that this is one of the pathways that contribute to transcellular HCO3 flux and fluid transport (8). We have examined this hypothesis by determining the effect of selective inhibition of the extracellular CA IV, and the consequent deletion of the putative CO2 gradient, on fluid transport. Benzolamide, a slowly permeant CA inhibitor, and a dextran-bound acetazolamide (mol wt 7,000) inhibit fluid transport by 28.8% and 22.8%, respectively (Fig. 5). Benzolamide has been reported to equilibrate slowly in human red blood cells at a rate of 7%/h (39) and thus might also act on intracellular CA. We therefore ran a series of experiments in which we initiated the inhibition of fluid transport and stromal swelling with benzolamide and then followed with a solution containing CA. This should lead to an immediate reduction of benzolamide to nanomolar levels and thus should return fluid transport to control levels. The swelling ceased immediately (Fig. 5), indicating that the action of benzolamide was extracellular, although corneal thickness did not return completely to control levels. Consistently, the effect of inhibition of CA IV on fluid transport shown here (
2030%) is significantly less than the known effect of inhibiting both CA IV and CA II, which was
4060% (12). In summary, slowly permeant or impermeant inhibitors of CA indeed reduce transendothelial fluid transport, but the inhibition is <30% of control (Fig. 5). In addition, as mentioned above, inhibition of Cl channels resulted in at most a 16% decrease of fluid transport (Fig. 4). It is interesting that the effects of impermeant and permeant CA inhibitors are additive. This is consistent with separate effects on an intracellular CA II and an extracellular CA IV, as discussed below.
In conclusion from the two segments above, from our evidence, inhibition of the two apical pathways previously proposed for anion efflux by Bonanno and colleagues, Cl channels (36, 43) and CO2 flux (6, 8), even if combined together, cannot account for more than 4045% of the baseline rate of transendothelial fluid transport. In the CO2 gradient hypothesis above (6, 8), the intracellular CA II would mediate HCO3 dehydration and the extracellular CA IV HCO3 hydration. However, the function of luminal and interstitial CA IV described for kidney proximal tubule (40) and for gastric parietal cells (39) is dehydration of HCO3, whereas the function of intracellular anhydrase is postulated to be hydration. In the scheme we propose below for the tandem of CAs in corneal endothelium, the enzymes function as proposed for the kidney and stomach.
Localization of NBC. We have demonstrated with confocal microscopy that in both cultured corneal endothelial cells and endothelial cells of freshly dissected cornea NBC proteins are located at the basolateral membrane and the apical membrane. As shown in Fig. 8, the density of staining at the apical membrane is significantly less than that at the basolateral membrane. This difference in the density of transport proteins is consistent with the HCO3 permeability of the apical membrane, which reportedly is four to three times lower than that of the basolateral membrane (8). If, in addition, the apical NBC transporter moves three molecules of HCO3 per turnover compared with two molecules per turnover of the basolateral NBC, then an apical protein density of 1722% of that in the basolateral membrane may be expected.
The distribution of NBC isoform(s) to both the basolateral and apical cell membranes reported here poses the question of how the cell can be polarized so as to generate vectorial HCO3 flux under such conditions. In this connection, NBC is distributed throughout the basolateral and apical membrane domains in cultured bovine endothelial cells and freshly dissected rabbit corneal preparations. Such distribution has also been observed recently in the ducts of the submandibular glands of the rat (32). However, in these duct cells there appears to be a gradient of distribution that changes from a purely basolateral localization in the proximal sections of the submandibular ducts to localization in both basolateral and apical membrane domains and then to apical localization only in the distal parts of the duct. In the corneal endothelium and in cultured bovine endothelial cells simultaneous basolateral and apical fluorescence is seen consistently in all cells, as shown in Figs. 68. Our finding is in contrast prima facie to the observation by Sun et al. (37) and Bok (5), who report that indirect fluorescence, due to antibody binding, is confined to the basolateral membrane. However, the analysis of the data was somewhat different. We used three-dimensional reconstruction of the confocal images to show the antibody distribution in cross sections of the cultured bovine endothelial cells, whereas Sun et al. (37) examined optical slices in the x-y plane only. In addition, we obtained cryosections of the freshly dissected corneal endothelium, again to visualize the antibody distribution in cell cross sections. Finally, there is a large difference in protein density between the apical and basolateral membrane domains, which might have led other investigators to consider the apical staining insignificant. For this and other reasons, it will be relevant to attempt to explore the putative function of the newly described apical NBC.
Thermodynamics of net transcellular HCO3 flux. From the preceding arguments, our results suggest that NBCs are expressed in both apical and basolateral membrane domains. Under these conditions, vectorial HCO3 transport across the endothelium via NBCs can occur either if different NBC isoforms are located on the apical and basolateral membranes or if the same isoform exists but functions differently. One of these isoforms would require a stoichiometry of 2 HCO3:1 Na+ or less for HCO3 influx across the basolateral membrane, whereas the other would require a stoichiometry of 3 HCO3:1 Na+ or greater for HCO3 efflux across the apical membrane. There is a conceivable alternative in which the NBC would function with the same 2:1 stoichiometry on both cell sides. In that case, HCO3 would be transported into the cell from both sides and high cell HCO3 could drive the reaction HCO3 + H+ > CO2 + H2O, with CO2 rapidly diffusing across the apical membrane. However, as mentioned above, for such vectorial diffusion of CO2 (Bonanno's hypothesis), there would have to be an apical extracellular CA IV reducing the extracellular CO2 by hydration; only thus could there be a basolateral-to-apical CO2 gradient and a net flow of CO2. Our results this far do not support that view; inhibition of an apical extracellular CA IV (Fig. 5) with the consequent removal of the hypothetical CO2 gradient reduces fluid transport by at most 29%. From the 16% inhibition of fluid transport (Fig. 4) seen in blocking apical anion channels, the expected inhibition of fluid transport, if due to CO2 flow, would be some 84% instead.
Figure 9 describes the thermodynamic limits of the operating range for a system consisting of NBCs with 2:1 and 3:1 stoichiometries. According to this figure, net HCO3 flux could take place over a relatively wide range of membrane potential and intracellular [HCO3] ([HCO3]i) values, namely 30 to 61 mV at 37 mM [HCO3]i and 2650 mM [HCO3]i at 45 mV membrane potential. These values fall within the physiologically expected range for an endothelial layer.
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An alternate transcellular anion transport model for corneal endothelial cells. On the basis of the data presented here, we propose a new model of transcellular anion transport across corneal endothelial cells (Fig. 10). In this model HCO3 enters the cell across the basolateral membrane via a NBC with a stoichiometry of 2 HCO3:1 Na+ (7). In parallel to this influx is a significantly smaller entry of Cl ions via the NKCC (9) and the anion exchanger. We propose that the anion efflux through the apical membrane is predominantly due to a NBC with a ratio of 1 Na+:3 HCO3 and a smaller Cl efflux through Cl channels. An apical NBC mediating HCO3 efflux is in line with evidence for Na+-HCO3 coupled exit cited above for cultured corneal endothelial cells (20). As Fig. 9 shows, a 3:1 NBC would operate relatively close to the thermodynamic limit. However, as hypothesized in Fig. 10, the combined action of the intracellular CA II and extracellular CA IV working in tandem would create/enhance a local [HCO3] gradient across the apical membrane to facilitate efflux across the NBC. The intracellular CA II would catalyze the conversion of CO2 to HCO3, whereas the membrane-bound extracellular CA IV would catalyze the reverse reaction. This is consistent with recent proposals that CA II is bound to the COOH terminal of NBC-1 in kidney proximal tubule (35); such binding apparently takes place only when the NBC is dephosphorylated and hence working in the 3:1 mode (15). There is also evidence that CA II and CA IV are tethered in close proximity to NBC extracellular and intracellular sites, respectively, in NBC-1-transfected HEK293 cells (3).
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GRANTS |
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FOOTNOTES |
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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.
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