Apical and basolateral CO2-HCOminus 3 permeability in cultured bovine corneal endothelial cells

Joseph A. Bonanno1, Yi Guan2, Sergey Jelamskii1, and Xiao Jun Kang2

1 School of Optometry, Indiana University, Bloomington, Indiana 47401; and 2 School of Optometry, University of California, Berkeley, California 94720


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Corneal endothelial function is dependent on HCO-3 transport. However, the relative HCO-3 permeabilities of the apical and basolateral membranes are unknown. Using changes in intracellular pH secondary to removing CO2-HCO-3 (at constant pH) or removing HCO-3 alone (at constant CO2) from apical or basolateral compartments, we determined the relative apical and basolateral HCO-3 permeabilities and their dependencies on Na+ and Cl-. Removal of CO2-HCO-3 from the apical side caused a steady-state alkalinization (+0.08 pH units), and removal from the basolateral side caused an acidification (-0.05 pH units). Removal of HCO-3 at constant CO2 indicated that the basolateral HCO-3 fluxes were about three to four times the apical fluxes. Reducing perfusate Na+ concentration to 10 mM had no effect on apical flux but slowed basolateral HCO-3 flux by one-half. In the absence of Cl-, there was an apparent increase in apical HCO-3 flux under constant-pH conditions; however, no net change could be measured under constant-CO2 conditions. Basolateral flux was slowed ~30% in the absence of Cl-, but the net flux was unchanged. The steady-state alkalinization after removal of CO2-HCO-3 apically suggests that CO2 diffusion may contribute to apical HCO-3 flux through the action of a membrane-associated carbonic anhydrase. Indeed, apical CO2 fluxes were inhibited by the extracellular carbonic anhydrase inhibitor benzolamide and partially restored by exogenous carbonic anhydrase. The presence of membrane-bound carbonic anhydrase (CAIV) was confirmed by immunoblotting. We conclude that the Na+-dependent basolateral HCO-3 permeability is consistent with Na+-nHCO-3 cotransport. Changes in HCO-3 flux in the absence of Cl- are most likely due to Na+-nHCO-3 cotransport-induced membrane potential changes that cannot be dissipated. Apical HCO-3 permeability is relatively low, but may be augmented by CO2 diffusion in conjunction with a CAIV.

bicarbonate permeability; epithelial transport; carbonic anhydrase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CORNEAL ENDOTHELIUM IS a thin monolayer of very "leaky" (transepithelial resistance of ~20 Omega  · cm2) epithelium covering the posterior surface of the cornea. Corneal endothelial cells regulate the movement of nutrients from the aqueous humor to the corneal stroma and surface epithelium, as well as movement of wastes back to the anterior chamber of the eye. The cornea is specialized in that it is a transparent optical structure. Transparency is determined by the regular spacing among collagen fibers, which is dependent on corneal hydration. The maintenance of corneal hydration is dependent on the endothelium, which provides most of the ion-coupled fluid transport activity in the cornea (18, 19). Thus damage to the endothelium by trauma, degeneration, or inflammation can lead to corneal edema and loss of transparency.

Endothelial ion and fluid transport is significantly slowed by carbonic anhydrase inhibitors (CAIs) or the removal of HCO-3 from the bathing solution (8, 10, 15, 23). This has generated interest in identifying HCO-3 transporters and understanding the role of carbonic anhydrase in HCO-3 transport. Previous studies have shown that endothelial cells in both cultured (4, 11, 14) and freshly isolated preparations (4) possess a potent Na+-dependent, DIDS-sensitive, electrogenic Na+-nHCO-3 cotransporter. Na+-nHCO-3 cotransport actively loads HCO-3 into endothelial cells and is the major intracellular pH (pHi) regulator during acid loads (4). Na+/H+ exchange is also present, but activity is very low at normal resting pHi (3). Cl- removal causes a DIDS-inhibitable HCO-3 influx in endothelial cells (4, 12). These fluxes are primarily due to membrane potential depolarization that secondarily causes an increase in Na+-nHCO-3 cotransport activity, with little or no contribution by Cl-/HCO-3 exchange (6). Thus the only significant confirmed HCO-3 transporter in the endothelium is the Na+-nHCO-3 cotransporter. HCO-3 could also be transported as CO2 in conjunction with membrane-bound carbonic anhydrase (CAIV) and cytosolic carbonic anhydrase (CAII) activity, as has been shown for the kidney (1, 22, 29). The CAI acetazolamide can slow HCO-3 influx and efflux in endothelial cells (5, 13); however, it is not clear whether this can be attributed partly or wholly to CAII (9) or CAIV activity (21, 27) in corneal endothelial cells.

To provide a model for transendothelial HCO-3 transport, the locations of the various transporters, channels, and carbonic anhydrase activities must be known. If a net HCO-3 flux were to contribute to corneal stroma (basolateral side) to aqueous humor (apical side) fluid transport and if Na+-nHCO-3 cotransport loads HCO-3 into the cells, then it is likely that this transporter will be located on the basolateral side. The nature of the apical efflux pathway, however, is more uncertain, especially because Cl-/HCO-3 exchange activity is weak. Using changes in pHi secondary to removing HCO-3 from apical or basolateral compartments, we set out to examine the relative apical vs. basolateral HCO-3 permeabilities and their dependencies on Na+ and Cl-. We also examined whether HCO-3 efflux across the apical membrane could be supplemented by CO2 diffusion and conversion to HCO-3 by CAIV.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Bovine corneal endothelial cells (BCEC) were cultured to confluence on glass coverslips or 13-mm AnoDisc (Whatman; Fisher Scientific) filters as previously described (3). Briefly, primary cultures from fresh cow eyes were established in T-25 flasks with 3 ml of DMEM, 10% bovine calf serum, and an antibiotic-antimycotic (100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml Fungizone); gassed with 5% CO2-95% air at 37°C, and fed every 2-3 days. These were subcultured to three T-25 flasks and grown to confluence in 5-7 days. The resulting second-passage cultures were then subcultured onto coverslips or filters, reaching confluence within 5-7 days. Cells were transferred to 1% serum-DMEM for at least 24 h before experiments.

Solutions and chemicals. The composition of the HCO-3- rich Ringer solution used throughout this study was (in mM) 150 Na+, 4 K+, 0.6 Mg2+, 1.4 Ca2+, 118 Cl-, 1 HPO2-4, 10 HEPES, 28.5 HCO-3, 2 gluconate-, and 5 glucose. Ringer solutions were equilibrated with 5% CO2, and pH was adjusted to 7.50 at 37°C. HCO-3-free Ringer (pH 7.5) was prepared by equimolar substitution of NaHCO3 with sodium gluconate. Low-HCO-3 Ringer (2.85 mM; pH 6.5) was prepared by replacing 25.65 mM NaHCO-3 with sodium gluconate. Cl--free Ringer was prepared by equimolar replacement of NaCl with sodium gluconate. Low-Na+ Ringer (10 mM) was prepared by replacement of 140 mM NaCl with 140 mM N-methyl-D-glucamine chloride. Osmolarity was adjusted to 300 ± 5 mosM with sucrose.

The 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was obtained from Molecular Probes (Eugene, OR). The CAI benzolamide (mol wt 320), which is known to be membrane impermeant during short-term exposure (20, 29, 30), was a generous gift from R. Hedges (Univ. of Washington). A polymer-linked CAI (POBUMS; mol wt 20,000) (17) was a generous gift from C. Conroy (Univ. of Florida). Cell culture supplies were obtained from GIBCO BRL (Grand Island, NY). CAII was obtained from Worthington (Indianapolis, IN). All other chemicals were obtained from Sigma (St. Louis, MO). Stock solutions of BCECF-AM (10 mM in DMSO) and nigericin (10 mM in ethanol) were stored desiccated at -20°C.

Perfusion. For independent perfusion of the apical and basolateral sides, a double-sided perfusion chamber was employed (see Ref. 6 for details). Cell-coated AnoDiscs were sandwiched between two thin (1 mm) plastic (Kel-F) plates, both of which had perfusion slots cut out at the center. Each perfusion slot (7 mm long × 3.1 mm wide) was connected to 23-gauge stainless steel tubing. The AnoDisc was placed in a 40-µm recess in the bottom Kel-F plate with the cells facing downwards. Round glass coverslips were seated on the outer surface of each Kel-F plate with a thin layer of vacuum grease to form apical and basolateral compartments (~22 µl). Stainless steel clamps on the outer surface of the plastic plates were screwed together sandwiching the AnoDisc firmly. The assembled chamber was placed on a water-jacketed (37°C) brass collar held on the stage of an inverted microscope (Diaphot; Nikon). The apical compartment faced the microscope objective (Zeiss; ×40, 1.2-mm working distance, 0.75 numerical aperture, water immersion). The apical and basolateral compartments were connected to separate sections of Phar-Med tubing, which, in turn, were connected to syringes placed in a Plexiglas warming box. Ringer solutions were placed in the syringes and maintained at 37°C. HCO-3-rich Ringer solutions were continually bubbled with 5% CO2. 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.

Measurement of pHi. pHi was measured with the pH-sensitive fluorescent dye BCECF (24). The cells were loaded by incubation in Ringer containing 1-5 µM BCECF-AM at room temperature for 30-60 min. Dye-loaded cells were then kept in Ringer for at least 30 min before use. Fluorescence excitation was provided by a 75-W xenon arc as part of a PTI ratio fluorescence system (Photon Technology, Monmouth Junction, NJ). The excitation wavelengths (495 and 440 nm) were obtained by passing the light through a DeltaRam monochromator. The excitation light was directed to the objective by a dichroic mirror centered at 505 nm. The fluorescence emission collected by the objective was passed through a barrier filter (540 ± 20 nm) and led to a photomultiplier for photon counting. Neutral-density filters (optical density 1-2) were included in the excitation path to minimize photobleaching. Synchronization of excitation with emission measurement and data collection was controlled by Felix software (Photon Technology). Fluorescence ratios were obtained at 1 s-1. The ratio of fluorescence emission to excitation at 495 nm to that at 440 nm (i.e., F495/F440) was calibrated against pHi by the high-K+-nigericin technique (3, 28). A calibration curve, which follows a pH titration equation, has been constructed for BCEC (3).

Immunoblotting. Fresh BCEC were scraped from dissected corneas, placed into ice-cold PBS containing a protease inhibitor cocktail (Complete; Boehringer Mannheim), and centrifuged at low speed for a brief period. Cell pellets were resuspended in 2% SDS sample buffer containing 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 5-10 min. An aliquot of the supernatant was taken for protein assay by the Bradford method (Bio-Rad). beta -Mercaptoethanol (5%) and bromphenol blue were added to the remainder of the supernatant, and the mixture was heated at 80°C for 4 min. The samples were applied to a 12% polyacrylamide gel with a 4.5% stacking gel (60 µg protein/lane). After electrophoresis at 20 mA, proteins were transferred to a polyvinylidene difluoride membrane overnight at 4°C. Membranes were incubated in PBS containing 5% nonfat dry milk for 1 h at room temperature and washed in PBS containing 0.05% Tween two to three times for 5 min. The blots were then incubated with anti-human CAIV primary antibody, kindly provided by W. Sly and A. Waheed (St. Louis Univ. School of Medicine). Next, the blots were washed four times with PBS-Tween, incubated with secondary antibody coupled to horseradish peroxidase (Sigma), and finally developed by enhanced chemiluminescence (DuPont). Films were scanned to produce digital images that were then assembled and labeled by using Microsoft PowerPoint software.

Data analysis. Initial slopes of pHi changes were taken from the first 20 s of data. Quantitative results are expressed as means ± SD. Student's t-test was used to determine significance (P < 0.05).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Two approaches were used to examine apical and basolateral HCO-3 permeabilities of cultured corneal endothelial cells. In the first approach, pHi was measured while cells were perfused in CO2-HCO-3-rich Ringer, followed by a brief exposure to CO2-HCO-3-free Ringer at the same pH. This is the constant-pH protocol. In this approach, pHi will be affected by both CO2 and HCO-3 fluxes. In the second approach, the test Ringer had reduced HCO-3 concentration ([HCO-3]) and pH, but the same CO2 concentration ([CO2]). This is the constant-CO2 protocol. In this approach, pHi will be affected by HCO-3 fluxes and the reduced Ringer pH. Both approaches, however, focus on the fact that net HCO-3 efflux should lead to a drop in pHi. Figure 1A shows the effect on pHi after removal of CO2-HCO-3 sequentially from the apical side and then the basolateral side by the constant-pH protocol. 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 (0.17 vs. 0.05 pH units) and was followed by a small acidification (-0.035 pH units). This acidification reflects HCO-3 efflux across the apical membrane. A new steady-state pHi was reached within 2-3 min and was always above the baseline pHi. When CO2-HCO-3-rich Ringer was reintroduced, there was a rapid acidification of the same magnitude as that of the initial alkalinization. This was followed by a recovery to the baseline pHi. On the basolateral side, Fig. 1A shows that after the initial alkalinization from CO2 efflux, there was a sharp acidification below the baseline (-0.15 pH units) and then a recovery to a new steady state ~0.05 pH units below the baseline. These data are summarized in Table 1. The smaller initial alkalinization and deeper and more rapid acidification on the basolateral side indicate that HCO-3 efflux is greater on the basolateral side than on the apical side. The concurrent CO2 and HCO-3 effluxes, however, make it difficult to quantitate these differences.


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Fig. 1.   Control intracellular pH (pHi) changes due to HCO-3 removal. A: constant-pH protocol. CO2-HCO-3-rich Ringer, pH 7.5, was replaced by HEPES-buffered CO2-HCO-3-free Ringer, pH 7.5, first on apical side only then on basolateral side. B: constant-CO2 protocol. Perfusate HCO-3 concentration ([HCO-3]) was reduced from 28.5 mM at pH 7.5 to 2.85 mM at pH 6.5, and both apical and basolateral solutions were gassed with 5% CO2. Apical and basolateral sides were exposed to low-[HCO-3] Ringer during periods indicated (boxes).


                              
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Table 1.   Effects on pHi during CO2-HCO-3 removal at constant pH

To remove the effects of CO2 fluxes seen in Fig. 1A, we next used the constant-CO2 protocol. Figure 1B shows a small drop in pHi (-0.06 pH units) when HCO-3 was removed from the apical side. However, on the basolateral side, HCO-3 removal caused an initial rapid drop (-0.21 pH units) followed by a small recovery. Table 2 summarizes these responses. In the presence of CO2-HCO-3, total buffering capacity (beta T) of corneal endothelial cells is ~55 mM/pH unit (3). By using the largest pH decrease from Table 2, the net equivalent effluxes (Delta pHi × beta T) from the apical side and basolateral side were calculated to be 2.8 and 11.6 mM, respectively. However, these flux values do not take into account the effect of reduced bath pH on the pHi change. To test this, we measured the drop in pHi due to changing the bath Ringer pH from 7.5 to 6.5 in CO2-HCO-3-free Ringer. The pHi dropped by 0.10 and 0.35 when the apical and basolateral pH values, respectively, were lowered. Taking into account the intrinsic (non-HCO-3) buffering capacity of these cells (10 mM/pH unit) (3), the net H+ influx values were 1.0 and 3.5 mM for the apical and basolateral sides, respectively. Thus ~64 and 70% of the initial pHi drop after removal of HCO-3 at constant CO2 from the apical and basolateral sides, respectively, are due to HCO-3 efflux. The corrected net HCO-3 effluxes were then 1.8 and 8.1 mM for apical and basolateral sides, respectively, indicating that the HCO-3 permeability of the basolateral side is more than four times that of the apical side. During the time it takes for the initial pHi drop to occur, however, other compensating pHi regulatory mechanisms may be activated. Therefore, we also calculated the initial HCO-3 flux on the basis of the initial -dpHi/dt, where t is time, again subtracting the initial -dpHi/dt due to changing the perfusate pH from 7.5 to 6.5. These results are shown in Table 3 and indicate that, on the basis of initial fluxes, the ratio of basolateral HCO-3 permeability to apical HCO-3 permeability is ~3.

                              
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Table 2.   Effects on pHi during HCO-3 removal at constant CO2


                              
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Table 3.   Apical and basolateral initial dpH/min and HCO -3 and H+ fluxes measured under constant-CO2 and HCO -3-free conditions

Na+ dependency. The high HCO-3 permeability of the basolateral side is most likely due to the Na+-nHCO-3 cotransporter. Therefore, reduced Na+ concentration ([Na+]) should have a greater effect on reducing HCO-3 flux on the basolateral side. Figure 2 shows a set of apical and basolateral responses at normal [Na+] (control) obtained by using the constant-pH protocol. The [Na+] was then reduced to 10 mM on both sides. This caused the pHi to drop ~0.2 units from 7.4 to 7.2, a drop due primarily to reversal of Na+-HCO-3 cotransport because Na+/H+ exchange is not active above pHi 7.15 (3). When apical CO2-HCO-3 was removed, cells became alkalinized as usual and came to a new steady state at a pHi similar to that of the control. In Fig. 2, it appears that the apical HCO-3 efflux rate was faster than the control rate. This was observed in two of four trials. However, when basolateral CO2-HCO-3 was removed, the HCO-3 efflux rate was slowed by 51 ± 10% (P < 0.05; n = 4). We conclude that basolateral HCO-3 efflux is diminished in low [Na+], but not apical flux, consistent with a Na+-nHCO-3 cotransporter located basolaterally.


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Fig. 2.   Effects of low Na+ concentration ([Na+]) on HCO-3 flux. Control apical (Ap) and basolateral (BL) CO2-HCO-3-free pulses were done by using constant-pH protocol. Perfusate [Na+] was then reduced on both sides to 10 mM, and apical and basolateral trials were repeated. Solid lines, slopes taken for HCO-3 efflux.

Cl- dependency. Figure 3 shows the apical and basolateral responses under the constant-pH protocol in the absence of Cl-. First, it should be noted that the resting pHi (7.45) is significantly higher than that in control Ringer (7.33). This has been shown previously and is due to NaHCO-3 influx via the transporter (Na+-nHCO-3 influx) secondary to membrane potential depolarization when Cl- is removed (6). When apical CO2-HCO-3 was removed in the absence of Cl-, after the initial alkalinization (+0.15 pH units) there was a significant decrease in pHi (-0.15 pH units), i.e., an increase in HCO-3 efflux, giving no net change in the steady-state pHi. Further, on the basolateral side, the initial alkalinization was slightly larger than that of the control (Fig. 1A) and the ensuing decrease in pHi was significantly diminished. However, the steady-state change in pHi was not significantly different from that for the control. These data are summarized in Table 1. Thus it appears that apical HCO-3 permeability is unmasked by the absence of Cl-. This may be due to release of a competitive efflux pathway or due to reduction in basolateral Na+-nHCO-3 cotransport activity, which is the most likely cause for the reduced basolateral efflux rate (see DISCUSSION).


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Fig. 3.   Effects of absence of Cl- on apical and basolateral HCO-3 fluxes. A: cells were perfused in Cl--free Ringer on both sides. This causes baseline pHi to rise to ~7.45. Apical and then basolateral CO2-HCO-3 was removed under constant-pH protocol. B: apical and then basolateral CO2-HCO-3 was removed under constant-CO2 protocol.

In an attempt to quantify the effect of the absence of Cl- on HCO-3 flux, Cl--free experiments were also done by using the constant-CO2 protocol. Figure 3B shows again the higher starting baseline pHi in the absence of Cl-. When HCO-3 was removed from the apical side, pHi went down ~0.04 units [0.02 ± 0.02 units (mean ± SD); n = 7] taking only ~10 s; then, within another 10 s, pHi was up 0.07 units [0.05 ± 0.03 (mean ± SD)]. Next the pHi decreased to a steady state that was 0.05 units below baseline. The initial rate of pHi decrease (i.e., HCO-3 efflux) was calculated from this last pHi decrease. Table 2 shows that the initial rate was not significantly different from the control rate. When HCO-3-rich Ringer was added back, pHi quickly went down 0.05 units [0.04 ± 0.02 units (mean ± SD)] and then rose 0.10 units [0.09 ± 0.02 units (mean ± SD)]. On the basolateral side, the initial decrease in pHi and the steady-state change in pHi were about the same as those for the control; however, there was a significant reduction (~30%) in the initial rate of decrease (Table 2). Thus the results from Fig. 3, A and B, indicate that basolateral HCO-3 flux is slowed in the absence of Cl-; however the net flux appears to be unaffected. The effect of Cl- on apical flux is more complex: an apparent increase in apical flux under constant-pH conditions yet no effect on net efflux under constant-pH conditions.

Apical CO2 flux. The steady-state alkalinization observed when apical CO2-HCO-3 is removed may indicate that apical CO2 efflux is a significant source of HCO-3, which could be generated by carbonic anhydrase at the surface of the apical membrane. Initially, we investigated the possibility of an extracellular CAIV by examining the effects of the polymer-linked CAI and benzolamide on CO2-HCO-3-induced changes in pHi by using cells cultured on coverslips. Figure 4A shows that CO2-induced acidification was slowed by 40% [45 ± 23% (mean ± SD); paired t-test; P < 0.05; n = 4] and that alkalinization when CO2 was removed was also slowed by 40% [24 ± 12% (mean ± SD); paired t-test; P < 0.05; n = 4] in the presence of 10 µM polymer-CAI. Figure 4B shows that 10 µM benzolamide slowed CO2-induced acidification by 45% [47 ± 13% (mean ± SD); n = 5] and slowed alkalinization on CO2 removal by 35% [30 ± 8% (mean ± SD); n = 5]. These results are consistent with the possibility that CO2 flux is influenced by an extracellular carbonic anhydrase.


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Fig. 4.   Effects of the extracellular carbonic anhydrase inhibitors (CAIs), polymer-CAI (poly-CAI) and benzolamide, on pHi transients secondary to adding or removing CO2-HCO-3-rich Ringer under constant-pH protocol in endothelial cells cultured on glass coverslips. A: cells were perfused in HCO-3-free Ringer and exposed briefly to CO2-HCO-3-rich Ringer as indicated (boxes). This was repeated in presence of 10 µM polymer-CAI. B: same as A except cells were exposed to 10 µM benzolamide as indicated.

We next focused on CO2 efflux across the apical membrane using the double-perfusion setup and benzolamide. Figure 5A shows that exposure to 1 µM apical benzolamide caused an immediate 0.05-pHi unit drop [-0.09 ± 0.04 (mean ± SD); n = 4]. Within 5 min, CO2-HCO-3 was removed from the apical side and the initial alkalinization rate and maximal alkalinization were reduced ~10%. Figure 5B shows a similar experiment with a 10-min exposure to 30 µM benzolamide. The alkalinization rate and maximal alkalinization were reduced by ~50%. Note that a brief washout could not reverse the inhibition. Figure 6 summarizes the dose effect of benzolamide on the initial rate and maximal alkalinization of the first test pulse, which occurred within 5 min of exposure to the drug. A longer washout of benzolamide was tried and yielded only limited reversibility. Figure 7A shows that after a 7-min exposure to 30 µM benzolamide, cells were washed for 30 min. Subsequent pulses showed only modest recovery of the initial rate (mean recovery ± SD = 12 ± 5%; n = 4) and amplitude (mean ± SD = 15 ± 13%).


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Fig. 5.   Effects of extracellular CAI benzolamide (Benz) on apical CO2 efflux-related changes in pHi under constant-pH protocol. A: effect of 1 µM apical benzolamide. B: effect of 30 µM benzolamide. Arrows indicate when CO2-HCO-3 was removed from apical side.



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Fig. 6.   Relationships between rate and amplitude (AMP) of initial alkalinization and dose of benzolamide. Constant-pH protocol was used as for Fig. 5. Data were taken from first CO2-HCO-3-free pulse, which usually occurred within 5 min of exposing cells to benzolamide.



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Fig. 7.   Effect of washout and exogenous carbonic anhydrase (CA) on recovery of benzolamide (Benz) inhibition. A: effect of 30-min washout on reversing benzolamide inhibition. Arrows indicate when CO2-HCO-3 was removed from apical side. B: effect of exogenous CA (5 mg/ml). At break in data cells were exposed to CA for 5 min, and then data collection was resumed.

Because release of benzolamide during washing appears to be slow, we attempted to reverse its effect by exposing cells to exogenous carbonic anhydrase (5 mg/ml). Figure 7B shows significant inhibition during a 7-min exposure to 30 µM benzolamide. Cells were then washed for ~20 min, showing modest recovery of the initial rate (mean recovery ± SD = 3 ± 2.5%; n = 3) and the maximal change in pH (mean ± SD = 5 ± 5%). Cells were then continually exposed to carbonic anhydrase. Subsequent pulses showed that inhibition was partially reversed (59 ± 30% recovery of the initial rate and 85 ± 20% recovery of the amplitude). These results show that extracellular carbonic anhydrase can partially reverse the inhibition of a brief exposure to benzolamide, indicating that benzolamide is acting primarily at the membrane.

Previous studies have indicated that CAIV activity is present in rabbit and mouse corneal endothelia (21, 27). To determine if the CAIV is present in the bovine corneal endothelium, we performed immunoblotting experiments with anti-human CAIV antibodies. The expected range for mammalian CAIV is 39-52 kDa (26). As shown in Fig. 8, the antibody detected a band at 45 kDa in both cultured and freshly isolated bovine corneal endothelia. A minor band at 40 kDa was more prominent in the cultured cells. A band at 27 kDa was observed in both preparations, but was much stronger in the fresh cells.


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Fig. 8.   Immunoblot of fresh and cultured bovine corneal endothelial cells by using anti-human CAIV antibodies. All lanes had 60 µg of protein. Lines at left indicate positions of molecular weight markers.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Two protocols, constant pH and constant CO2, were used to examine HCO-3 permeability in corneal endothelial cells. When the constant-pH protocol is used, the relative steady-state pH change after CO2-HCO-3 removal can give a general impression of the relative apical and basolateral permeabilities. Figure 1A clearly shows greater basolateral HCO-3 efflux. The initial pHi changes, however, are due to CO2 fluxes, thus making quantitative comparisons of HCO-3 flux difficult. The constant-CO2 protocol eliminates the CO2 fluxes, but introduces a low bath pH, for which compensation is needed. The exposure to low bath pH did not increase the rate of BCECF dye leakage from cells, indicating that the brief unilateral exposure to low pH was not detrimental. Using the initial HCO-3 flux yields a basolateral permeability that is about threefold greater than the apical permeability (Table 3). This is probably a low estimate because no compensation was made for the AnoDisc membrane itself, which adds another basal diffusion barrier.

Na+ dependency. Previous studies have shown that the corneal endothelial cells possess a Na+-dependent, DIDS-sensitive HCO-3 cotransporter that is sensitive to membrane potential (4, 11, 14) and that will raise cytosolic [Na+] when exposed to HCO-3 (4). On the basis of the steady-state levels of intracellular and extracellular [HCO-3] and [Na+] and the average membrane potential of endothelial cells, we concluded that this cotransporter would have a Na+-HCO-3 stoichiometry of ~1:2 and would act as a HCO-3 uptake system (4). Application of the anion transport inhibitor DIDS to the basolateral side produced cell acidification, whereas apical exposure had a more variable effect (6). From these studies, it was concluded that a Na+-nHCO-3 cotransporter is located on the basolateral side. This conclusion is consistent with our finding, shown in Fig. 2, that basolateral HCO-3 fluxes and not apical fluxes are slowed in low [Na+].

Cl- dependency. Cl- has been shown to be essential for fluid transport by the corneal endothelium (31). A recent examination of cultured corneal endothelial cells for Cl-/HCO-3 exchange showed little to no anion exchange activity (6), and there is no evidence for other types of Cl--dependent HCO-3 transporters. However, anion channel activity has been demonstrated, and altering bath Cl- concentration ([Cl-]) can have profound effects on endothelial membrane potential, which in turn will secondarily affect Na+-nHCO-3 cotransport flux. Further, a limited amount of HCO-3 permeability through Cl- channels can be demonstrated in the form of cell alkalinization in the absence of Na+ in response to cAMP activation of anion permeability (2). Figure 3, A and B, shows that basolateral HCO-3 efflux is slowed in the absence of Cl-. This may indicate a Cl- dependency for Na+-nHCO-3 cotransport; however previous studies have shown no such dependency in cells grown on coverslips or in freshly isolated cells (4). Further, Tables 1 and 2 indicate that the net change in pHi was the same in the absence of Cl- as in the control, arguing that the efflux rate is reduced, but not the net flux. The most likely explanation is that the absence of Cl- slows the dissipation of membrane potential changes during Na+-nHCO-3 flux. For example, during Na+-nHCO-3 influx the membrane potential hyperpolarizes, slowing further Na+-nHCO-3 influx. The hyperpolarization could be partially offset by Cl- efflux through anion channels, because Cl- is above its electrochemical equilibrium (5). However, in the absence of Cl-, this depolarization cannot be offset by Cl- and HCO-3 flux is therefore slowed. Further studies are needed to investigate this possibility.

The effect of the absence of Cl- on apical HCO-3 flux was more complex. Under the constant-pH protocol, apical HCO-3 net efflux was greater than control efflux (Fig. 3A; Table 1). This is the opposite of what is expected for an apical Cl-/HCO-3 exchanger, but might be explained by the presence of apical anion channels that have some HCO-3 permeability (2, 6, 7). If the inherent permeability of an apical channel to Cl- is higher than its permeability to HCO-3, then this together with the higher bath and cytoplasmic [Cl-] under control conditions would limit HCO-3 access to the channel. However, when Cl- is absent, this competition is removed and greater HCO-3 flux can occur. Another possibility, as explained above, is that Na+-nHCO-3 cotransport activity is slowed, which allows the limited apical HCO-3 efflux to have a greater effect on pHi. Under the constant-CO2 protocol, the initial rate of HCO-3 efflux appeared to be the same as the control rate. However, this is difficult to know for certain because the pHi first went down quickly (-0.02 pH units), then up quickly (+0.05 pH units), then down again at a rate comparable to the control rate. When HCO-3 was added back, pHi quickly went down, then rose 0.1 units back to the baseline. These transient changes cannot be due to CO2 fluxes, but possibly they are due to small membrane potential changes. If apical anion channels with HCO-3 permeability are present, then apical HCO-3 removal would cause a small depolarization, increasing Na+-nHCO-3 cotransport flux transiently, which could explain the transient increase in pHi. When apical HCO-3 is added back, a small hyperpolarization could take place, transiently depressing Na+-nHCO-3 cotransport and causing the transient decrease in pHi. Again, these transients are not observed in the controls because the presence of Cl- would limit the effect of changing apical [HCO-3] on the membrane potential. Clearly, these possibilities will require further testing.

Apical CO2 flux. When CO2-HCO-3 is removed there is an initial alkalinization due to the rapid efflux of CO2. The rate and extent of this initial response are influenced by the concurrent rate of HCO-3 efflux. For example, a small alkalinization is observed when basolateral CO2-HCO-3 is removed because the concurrent HCO-3 efflux is large. The opposite response, a high sustained alkalinization, is observed on the apical side, indicating that CO2 efflux exceeds HCO-3 efflux. Thus a significant component of apical HCO-3 flux may be in the form of CO2, which then could be converted rapidly to HCO-3 by a CAIV. Inhibiting the conversion of CO2 to HCO-3 at the membrane can reduce the local CO2 diffusion gradient (16, 25) and thus slow CO2 flux. If CO2 efflux is slowed, then the rate and extent of alkalinization will be reduced because HCO-3 efflux will have a proportionally greater effect on pHi. Both the polymer-linked CAI and benzolamide significantly reduced the rate and extent of pHi change in endothelial cells cultured on coverslips when CO2-HCO-3 was removed (Fig. 4), indicating that CO2 fluxes can be influenced by a CAIV. Similarly, when apical CO2 efflux was examined in the presence of benzolamide, the initial rate and extent of alkalinization were significantly reduced (Figs. 5-7). Furthermore, as previously noted for muscle (25), the reversibility of benzolamide inhibition was small even after 30 min of washout. We used exogenous carbonic anhydrase (5 mg/ml) in an attempt to restore the membrane activity (or possibly scavenge bound benzolamide) as was shown for the kidney (29). Exposure to carbonic anhydrase restored 59% of the initial alkalinization rate and 85% of the total alkalinization (Fig. 7B). These results are consistent with benzolamide, acting primarily at the membrane, inhibiting a carbonic anhydrase that enhances CO2 diffusion across the membrane.

Initial immunofluorescence reports indicated that only CAII and not CAIV was associated with the corneal endothelium (9). However, more recently, strong corneal endothelial apical membrane-associated carbonic anhydrase activity has been demonstrated histochemically in the CAII-deficient mouse (21) and in the normal rabbit (27). We used Western blotting to determine if CAIV immunoreactivity was present in the endothelial cells. Positive bands for both cultured and freshly isolated cells (40-45 kDa) were observed in the correct range (39-52 kDa) for mammalian CAIV (26). A strong band was also seen at 27 kDa for the fresh cells. This may be related to the mechanical scraping used to collect the fresh tissue. Together with some unavoidable proteolysis, the scraping may yield more fragmentation. Taken together, the immunoblot results, polymer-CAI data, benzolamide data, and the demonstrated histochemical activity at the apical membrane strongly suggest that CAIV is present on the apical membrane. Similar types of physiological experiments can be used to determine if a basolateral CAIV could be present and could increase the availability of HCO-3 for Na+ and HCO-3 uptake.

In summary, the permeability of the corneal endothelium to HCO-3 is at least three times greater for the basolateral membrane than for the apical membrane. The Na+-nHCO-3 cotransporter, located on the basolateral membrane, provides robust uptake of HCO-3. Na+-nHCO-3 cotransport is slowed in the absence of Cl-, most likely because of changes in membrane potential that cannot be dissipated. Because of the low apical HCO-3 permeability, intracellular [HCO-3] builds up and HCO-3 is converted to CO2 by CAII. The accumulated CO2 could augment apical HCO-3 flux given the presence of CAIV on the apical membrane.


    ACKNOWLEDGEMENTS

This work was supported by National Eye Institute Grant EY-08834.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. A. Bonanno, Indiana Univ., School of Optometry, 800 E. Atwater Ave., Bloomington, IN 47401 (E-mail: jbonanno{at}indiana.edu).

Received 8 April 1999; accepted in final form 1 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 277(3):C545-C553
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