Apical and basolateral
CO2-HCO
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 |
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 |
THE CORNEAL ENDOTHELIUM IS a thin monolayer of very
"leaky" (transepithelial resistance of ~20
· 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 |
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).
-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 |
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).
|
|
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 (
T)
of corneal endothelial cells is ~55 mM/pH unit (3). By using the
largest pH decrease from Table 2, the net equivalent effluxes
(
pHi ×
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 3.
Apical and basolateral initial dpH/min and
HCO 3 and
H+ fluxes measured under constant-CO2
and HCO 3-free conditions
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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.
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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.
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|
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.
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|
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.
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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 |
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.
 |
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