Expression, localization, and functional evaluation of CFTR
in bovine corneal endothelial cells
Xing Cai
Sun and
Joseph A.
Bonanno
Indiana University School of Optometry, Bloomington,
Indiana 47405
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ABSTRACT |
HCO
-dependent
fluid secretion by the corneal endothelium controls corneal hydration
and maintains corneal transparency. Recently, it has been shown that
mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the corneal endothelium; however, protein expression, functional localization, and a possible role in HCO
transport have not been reported. Immunoblotting for CFTR showed a
single band at ~170 kDa for both freshly isolated and primary cultures of bovine corneal endothelial cells. Indirect
immunofluorescence confocal microscopy indicated that CFTR locates to
the apical membrane. Relative changes in apical and basolateral
chloride permeability were estimated by measuring the rate of
fluorescence quenching of the halide-sensitive indicator
6-methoxy-N-ethylquinolinium iodide during Cl
influx in the absence and presence of forskolin (FSK). Apical and
basolateral Cl
permeability increased 10- and 3-fold,
respectively, in the presence of 50 µM FSK. FSK-activated apical
chloride permeability was unaffected by H2DIDs (250 µM);
however, 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB; 50 µM) and glibenclamide (100 µM) inhibited activated Cl
fluxes by 45% and 30%, respectively. FSK-activated basolateral Cl
permeability was insensitive to NPPB, glibenclamide,
or furosemide but was inhibited 80% by H2DIDS.
HCO
permeability was estimated by measuring changes
in intracellular pH in response to quickly lowering bath
[HCO
]. FSK (50 µM) increased apical
HCO
permeability by twofold, which was inhibited
42% by NPPB and 65% by glibenclamide. Basolateral
HCO
permeability was unaffected by FSK. Genistein
(50 µM) significantly increased apical HCO
and
Cl
permeability by 1.8- and 16-fold, respectively. When
50 µM genistein was combined with 50 µM FSK, there was no further
increase in Cl
permeability; however,
HCO
permeability was reduced to the control level.
In summary, we conclude that CFTR is present in the apical membrane of
bovine corneal endothelium and could contribute to transendothelial
Cl
and HCO
transport. Furthermore,
there is a cAMP-activated Cl
pathway on the basolateral
membrane that is not CFTR.
cornea; endothelium; chloride permeability; MEQ; bicarbonate
permeability; intracellular pH; BCECF; forskolin; cAMP; genistein
 |
INTRODUCTION |
CORNEAL TRANSPARENCY
and thus good vision are dependent on the hydration of the corneal
stromal connective tissue. When the stroma becomes edematous (i.e.,
tissue hydration >78%), there is increased light scatter from
collagen fibers, which degrades the retinal image and gives the cornea
a hazy appearance. The glycosaminoglycans of the stroma exert a fluid
imbibition pressure or "leak" that must be opposed by a cellular
ion "pump" to control corneal hydration. Damage to the anterior
corneal epithelium or posterior corneal endothelium can produce stromal
edema; however, it is the endothelial cells, a thin monolayer of
"leaky epithelium," that provides most of the ion-coupled fluid
transport activity or pump function for the cornea (32).
Thus disorders of the corneal endothelium (e.g., Fuchs' endothelial
dystrophy) produce corneal edema and loss of vision (1).
Corneal endothelial fluid transport is dependent on the presence of
both HCO
(11, 16, 36) and
Cl
(50). In addition, fluid transport is
slowed by stilbene derivatives (26, 50) and carbonic
anhydrase inhibitors (17, 23, 36). More recently, it has
been shown that bumetanide can induce corneal edema (22),
indicating roles for both Cl
and HCO
transporters. HCO
and Cl
are loaded
into corneal endothelial cells, to levels above electrochemical equilibrium (5), on the basolateral (stromal) side by the
Na+-2HCO
cotransporter (NBC-1)
(45) and the
Na+-K+-2Cl
cotransporter (NKCC1)
(20, 22), respectively. What is less clear, however, is
the mechanism for apical anion efflux.
Three possible mechanisms for HCO
secretion across
the apical membrane of corneal endothelial cells have been postulated:
1) Cl
/HCO
exchange,
2) CO2 efflux and conversion to
HCO
by a membrane-bound carbonic anhydrase (CAIV),
and 3) conductive flux via anion channels. The anion
exchanger (AE2) is expressed in freshly isolated corneal endothelial
cells (7, 46); however, its apical or basolateral localization has not been determined. Primary cultures of bovine corneal endothelial cells (BCEC) do not express AE2 (46)
and show little if any anion exchange activity (7).
Nevertheless, cultured endothelial cells can transport fluid at the
same level as the complete cornea (34), indicating that
anion exchange may not have a role in fluid secretion. Apical membrane
CAIV has been shown to enhance apical CO2 diffusion
(4); however, whether this can significantly contribute to
transendothelial HCO
flux has not been demonstrated.
Corneal endothelial fluid transport is stimulated by increasing
cytosolic [cAMP] (12, 37). Furthermore, cAMP activates
Cl
transport in cultured corneal endothelial cells
(5), and recently it has been shown that mRNA for the
cystic fibrosis (CF) transmembrane regulator (CFTR) is present in the
corneal endothelium (46). Because CFTR has significant
permeability to HCO
as well as to Cl
(8, 35, 44), it could serve as a possible apical anion efflux pathway.
In this study, we examine CFTR protein expression in fresh and cultured
corneal endothelial cells. We used primary cultures of endothelial
cells to determine the physical and functional localization of CFTR. We
show that CFTR protein is expressed and exclusively locates to the
apical membrane. This localization corresponds with forskolin
(FSK)-stimulated Cl
and HCO
fluxes
being inhibited by 5-nitro-2-(3-phenylpropyl-amino)benzoic acid
(NPPB) and glibenclamide only on the apical membrane.
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MATERIALS AND METHODS |
Cell culture.
BCEC were cultured to confluence onto 13-mm Anodiscs or T-25 flasks as
previously described (6, 29). Briefly, primary cultures
from fresh cow eyes were established in T-25 flasks with 3 ml DMEM,
10% bovine calf serum, and antibiotic-antimycotic (100 U/ml
penicillin, 100 U/ml streptomycin, and 0.25 µg/ml Fungizone) gassed
with 5% CO2-95% air at 37°C and fed every 2-3
days. Primary cultures were subcultured to three T-25 flasks and grown
to confluence in 5-7 days. The resulting second passage cultures
were then further subcultured onto Anodisc membranes and allowed to
reach confluence within 2 wk.
Immunoprecipitation.
Fresh BCEC were scraped from dissected cow corneas that had been kept
on ice for 2-3 h since death. The cell scrapings were placed into
ice-cold PBS containing a protease inhibitor cocktail (Complete,
Boehringer-Mannheim) and washed twice. Cell pellets were obtained by
low-speed centrifugation and resuspended in immunoprecipitation (IP)
buffer [1.0% Nonidet P-40, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 50 mM Tris · HCl (pH 8.0) containing a
protease inhibitor cocktail]. Cultured cells in T-25 flasks were
washed with PBS and dissolved directly in IP buffer. Both preparations were sonicated on ice. Sonicated samples were centrifuged at 10,000 g for 10 min at 4°C. The supernatant was transferred and
then incubated for 16-18 h with a monoclonal antibody (2 µg
antibody · mg protein
1 · ml IP
buffer
1) directed against the COOH terminus of CFTR (R&D
Systems; Minneapolis, MN). Immobilized protein A agarose was added to
the solution during the final 2 h of incubation. The immune
complexes were collected by centrifugation at 10,000 g for
15 s at 4°C and washed three times with ice-cold IP buffer (1 ml). The immune complexes were resuspended with 50 µl Laemmli sample
buffer [2% SDS, 10% glycerol, 100 mM dithiothreitol, 60 mM Tris (pH
6.8), and 0.01% bromophenol blue] and heated to 80°C for 10 min
before loading. After being separated by 8% SDS-PAGE, samples were
transferred to a polyvinylidene fluoride membrane. The membrane was
blocked with 5% nonfat dry milk for 1 h at room temperature and
then probed with the anti-CFTR antibody (1:1,000) in PBS containing 5%
nonfat dry milk for 1 h at room temperature with shaking. Next,
the blots were washed five times for 5 min each with PBS-Tween 20, incubated with goat anti-mouse secondary antibody coupled to
horseradish peroxidase (Sigma) for 1 h at room temperature, washed
with PBS-Tween 20 five times for 5 min each, and then developed by
enhanced chemiluminescence. Films were scanned to produce digital
images that were then assembled and labeled using Microsoft Powerpoint software.
Immunofluorescence and confocal microscopy.
Cultured cells grown to confluence on coverslips were washed three to
four times with PBS and fixed for 30 min in PLP fixation solution [2%
paraformaldehyde, 75 mM lysine, 10 mM sodium periodate, and 45 mM
sodium phosphate (pH 7.4)] on a rocker. After fixation, the cells were
washed three to four times with PBS. Coverslips were then kept in PBS
for 20 min containing 0.01% saponin to permeablize the cell membranes
and washed three times in PBS. Cells were blocked for 1 h in PBS
containing 0.2% BSA, 5% goat serum, 0.01% saponin, and 50 mM
NH4Cl. To aid in CFTR membrane localization, indirect double immunofluorescence staining for CFTR and ZO-1 was performed. A
mixture of mouse anti-human monoclonal CFTR antibody diluted 1:10 and
rat anti-ZO-1 monoclonal antibody diluted 1:100 in PBS-goat serum (1:1)
was applied onto coverslips at room temperature for 1 h.
Coverslips were washed three times for 15 min in PBS containing 0.01%
saponin. The mixture of secondary antibodies conjugated to Oregon green
(CFTR) and Texas red (ZO-1) (1:500, Molecular Probes; Eugene, OR) was
then applied for 1 h at room temperature. Coverslips were washed
and mounted with Prolong anti-fade medium according to the
manufacturer's (Molecular Probes) instructions. Fluorescence was
observed at ×40 with a standard epifluorescence microscope equipped
with a cooled charge-coupled device camera. Fluorescence of selected
specimens was documented with a Bio-Rad laser scanning confocal
microscope to determine membrane localization.
Microscope perfusion.
For measurement of Cl
and HCO
fluxes
using fluorescent dyes, cells cultured to confluence on Anodisc membranes were placed in a double-sided perfusion chamber designed for
independent perfusion of the apical and basolateral sides as previously
described (7). The chamber was placed on a water-jacketed (37°C) brass collar held on the stage of an inverted microscope (Nikon Diaphot) and viewed with a long working distance (1.2 mm) water
immersion objective (×40, Zeiss). Apical and basolateral compartments
were connected to hanging syringes containing Ringer solutions in a
Plexiglas warming box (37°C) using Phar-Med tubing. The flow of the
perfusate (~0.5 ml/min) was achieved by gravity. Two independent
eight-way valves were employed to select the desired perfusate for the
apical and basolateral chambers.
Measurement of intracellular [Cl
].
Relative intracellular [Cl
] changes in cultured BCEC
were assessed with the halide-sensitive fluorescent dye
6-methoxy-N-ethylquinolinium iodide (MEQ). Corneal
endothelial cells on Anodiscs were exposed to the nonfluorescent
cell-permeant reduced quinoline derivative of MEQ (diH-MEQ) (3,
51), which is oxidized to MEQ within the cytoplasm. Cells were
exposed to 10 µM diH-MEQ for 10 min at room temperature in
Cl
-free Ringer solution and washed for 30 min with
Cl
-free Ringer solution. Cellular fluorescence was
measured with a microscope spot fluorimeter (DeltaRam, Photon
Technology International; Monmouth Junction, NJ). Fluorescence was
excited at 365 ± 10 nm and emission was collected at 420-450
nm. Synchronization of excitation with emission measurement and data
collection (1 s
1) were controlled by Felix software
(PTI). Relative differences in Cl
permeability between
control and experimental conditions in the same cells were determined
by comparing the percent change in MEQ fluorescence (F/F0)
after addition of Cl
to either the apical or basolateral
bath, where F0 is the fluorescence in the absence of
Cl
. The maximum slope of the fluorescence change was
determined by calculating the first derivative using Felix software.
Measurement of intracellular pH.
BCEC cultured onto permeable Anodisc filters were loaded with the
pH-sensitive fluorescent dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)
by incubation in HCO
-free Ringer solution containing
1-5 µM BCECF-AM at room temperature for 30-60 min.
Dye-loaded cells were then kept in HCO
-free Ringer
solution for at least 30 min before use. Fluorescence was excited
alternately at 495 ± 10 and 440 ± 10 nm at 1 ratio
(F495/F440) s
1, and ratios were
calibrated against intracellular pH (pHi) by the high
K+-nigericin technique (47). Anodiscs were
perfused on both sides with HCO
-rich Ringer (BR)
solution. Apical or basolateral HCO
efflux was then
induced by introduction of a low-HCO
Ringer (LB)
solution. This was then repeated in the presence of agonists and
inhibitors. The maximum slope of the change in pHi over
time (dpHi/dt) after introduction of LB solution
was determined by calculating the first derivative using Felix
software. Data are expressed as means ± SE. Student's
t-test was used to determine significance (P < 0.05).
Solutions and chemicals.
The composition of the BR solution used throughout this study was (in
mM) 150 Na+, 4 K+, 0.6 Mg2+, 1.4 Ca2+, 118 Cl
, 1 HPO
, 10 HEPES, 28.5 HCO
, 2 gluconate
, and 5 glucose. Ringer solutions were equilibrated with 5% CO2, and pH was adjusted to 7.50 at 37°C. LB solution (2.85 mM, pH 6.5)
was prepared by replacing 25.65 mM NaHCO3 with sodium
gluconate. Cl
-rich, HCO
-free Ringer
solution (pH 7.5) was prepared by equimolar substitution of
NaHCO3 with sodium gluconate. Cl
-free Ringer
solution was prepared by equimolar replacement of NaCl and KCl with
sodium nitrate and potassium nitrate. In some experiments, gluconate
salts were used. Osmolarity was adjusted to 295 ± 5 mosM with sucrose.
MEQ, BCECF-AM, and H2DIDS were obtained from Molecular
Probes. FSK and genistein were obtained from LC Laboratories (Woburn, MA). Glibenclamide and NPPB were from Sigma (St. Louis, MO).
 |
RESULTS |
IP and indirect immunofluorescence.
To demonstrate the expression of CFTR protein in bovine corneal
endothelium, CFTR was immunoprecipitated from cultured and fresh BCEC
lysates with mouse anti-human CFTR antibody. Figure 1 shows that the CFTR antibody produced
strong positive bands for both cultured and fresh corneal endothelium
at ~170 kDa, which is the expected range for mature CFTR
(30). Further evidence for the expression of CFTR in BCEC
is provided by indirect immunofluorescence confocal micrographs, as
shown in Fig. 2. Cultured BCEC were
stained for both CFTR and the tight junction protein ZO-1. CFTR
fluorescence was apparent just apical to ZO-1 and at the same level as
ZO-1, but not basolateral to ZO-1. This result indicates that CFTR is predominately located at the apical membrane of BCEC.

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Fig. 1.
CFTR immunoprecipitation analysis of cultured (CBCEC) and
fresh bovine corneal endothelial cells (FBCEC). CFTR was precipitated
by mouse anti-human CFTR monoclonal antibody and protein A-linked
agarose beads from whole cell extracts. The immunoprecipitates were
separated by polyacrylamide gel electrophoresis, transferred to
polyvinylidene fluoride membranes, and then probed with an antibody to
CFTR.
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Fig. 2.
Laser scanning confocal immunofluorescence microscopy of
ZO-1 (Texas red-linked secondary antibody) and CFTR-stained (Oregon
green-linked secondary antibody) cultured bovine corneal endothelium.
The 6 images are sequential from the most basolateral section
(A) to the most apical (F). The z-axis
separation between images is 0.5 µm. Sections more basal to
F did not contain fluorescence.
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cAMP increases apical and basolateral Cl
permeability.
If CFTR contributes to Cl
permeability and is localized
to the apical membrane in BCEC, then increasing cellular cAMP should enhance apical Cl
permeability. This was tested by
measuring the relative change in MEQ fluorescence quenching due to
Cl
influx in the absence and then presence of FSK (Fig.
3). Both apical and basolateral sides
were initially perfused with Cl
-free (nitrate
substituted) Ringer solution. When Cl
was added to the
apical side for 90 s, Cl
entry caused a small slow
decrease in MEQ fluorescence (1.6% min
1). Immediately
after this 90-s exposure to Cl
, 50 µM FSK was
introduced in the continued presence of Cl
for ~1 min.
As shown in Fig. 3, this dramatically accelerated the decrease in MEQ
fluorescence by ~11-fold (16.3% min
1) relative to the
control, indicating that cAMP produced a significant increase in apical
Cl
permeability. The same procedure was then
performed on the basolateral side to test whether cAMP could enhance
basolateral Cl
permeability. After a 10-min wash with
Cl
-free Ringer solution on the apical side,
Cl
was introduced for 90 s on the basolateral side.
This caused a relatively faster and larger decrease in MEQ fluorescence
(3.3% min
1) than on the apical side. This is consistent
with previous studies and is contributed primarily by the basolateral
Na+-K+-2Cl
cotransporter (NKCC1)
(20, 22). The application of 50 µM FSK also accelerated
the decrease in MEQ fluorescence during basolateral Cl
influx by 2.7-fold (9% min
1) relative to the
controls. Similar results were obtained if the sequence of
Cl
addition/FSK exposure was first basolateral and then
followed by apical, indicating that the ~15-min wash between FSK
pulses was sufficient time to reduce cAMP to control levels. Figure
3B summarizes the results, indicating that apical and
basolateral Cl
permeability was increased by FSK ~10-
and 3-fold, respectively.

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Fig. 3.
Changes in 6-methoxy-N-ethylquinolinium iodide
(MEQ) fluorescence (F/F0) after apical or basolateral
Cl addition in the absence and presence of forskolin
(FSK). BCEC were cultured to confluence on Anodisc permeable membranes.
They were loaded with MEQ in the absence of Cl (nitrate
substituted), washed, and placed in a microscope-fluorimeter two-sided
perfusion chamber. A: both apical and basolateral
compartments were initially perfused with Cl -free
(nitrate substituted) Ringer solution. Arrows indicate when
Cl -rich Ringer solution and 50 µM FSK were applied from
the apical or basolateral side. B: percent change in maximum
F/F0 (%dF/F0) per minute. Values are
means ± SE. *Significantly different from controls (Con;
n = 6, P < 0.05).
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If the changes in Cl
permeability induced by FSK on the
apical and basolateral sides were contributed by activation of CFTR, then the activated Cl
fluxes should be sensitive to the
Cl
channel inhibitors NPPB and glibenclamide, which are
known (in some systems) to inhibit CFTR, but should not be inhibited by DIDS. We found that H2DIDS (200 µM) had no effect on the
FSK-activated apical Cl
flux but did reduce FSK-activated
basolateral flux by 80 ± 20% (n = 4, data not
shown). Conversely, Fig. 4A
shows that when Cl
was added on the apical side in the
presence of 50 µM FSK and 50 µM NPPB, the rate of MEQ fluorescence
quenching was reduced by ~45% relative to FSK alone. On the other
hand, Fig. 4B shows that when Cl
was
introduced on the basolateral side in the presence of 50 µM FSK and
50 µM NPPB, the acceleration in the decrease of MEQ fluorescence
caused by FSK did not change. These results are summarized in Fig.
4C, which shows that NPPB significantly reduced
FSK-activated Cl
permeability on the apical side but not
on the basolateral side.

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Fig. 4.
Effects of 5-nitro-2-(3-phenylpropyl-amino)benzoic acid
(NPPB) on FSK-activated Cl flux on the apical or
basolateral side. A: effect of NPPB on the apical side. Both
apical and basolateral compartments were initially perfused with
Cl -free (nitrate substituted) Ringer solution as in Fig.
3. Arrows indicate the applications of Cl -rich Ringer
solution and 50 µM FSK on the apical side. Before the addition of
NPPB, there was a 10-min wash with Cl -free Ringer
solution on the apical side. B: effect of NPPB on the
basolateral side. C: maximum %dF/F0 per minute.
Values are means ± SE. #Significantly less than FSK alone
(n = 5, P < 0.05).
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Figure 5A shows the effect of
glibenclamide on FSK-activated Cl
flux at the apical
side. When Cl
was applied on the apical side in the
presence of 50 µM FSK together with 100 µM glibenclamide, the rate
of MEQ fluorescence quenching was reduced by ~30% relative to FSK
alone. On the other hand, Fig. 5B shows that on the
basolateral side the addition of glibenclamide did not cause any
inhibition in the rate of fluorescence change induced by FSK. These
results are summarized in Fig. 5C, which shows that
glibenclamide, like NPPB, inhibited FSK-activated Cl
flux
only on the apical side.

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Fig. 5.
Effects of glibenclamide (Glib) on FSK-activated
Cl flux on the apical or basolateral side. A:
effect of Glib on the apical side. Both apical and basolateral
compartments were initially perfused with Cl -free
(nitrate substituted) Ringer solution as in Figs. 3 and 4. Arrows
indicate Cl introduction and FSK addition. Before the
addition of Glib, there was a 10-min wash with Cl -free
Ringer solution on the apical side. B: effect of Glib on the
basolateral side. C: maximum %dF/F0 per minute.
Values are means ± SE. #Significantly less than FSK alone
(n = 8, P < 0.05).
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The Na+-K+-2Cl
cotransporter
(NKCC1) provides significant basolateral Cl
permeability
in BCEC (20, 22). In some systems, NKCC1 can be activated
by cAMP (10, 13, 15, 27, 31, 41), so we tested if
FSK-activated basolateral Cl
permeability could be
provided by NKCC1. We found that 100 µM furosemide (bumetanide was
not used due to its fluorescence at 360 nm), which strongly inhibits
basolateral Na+-K+-2Cl
cotransport in endothelial cells (20), had no effect on
the basolateral FSK-activated Cl
flux (n = 4, data not shown).
In summary, FSK-activated apical Cl
permeability was
inhibited by NPPB and glibenclamide but not H2DIDS,
consistent with CFTR having an apical location. On the other hand,
FSK-activated basolateral Cl
permeability was inhibited
by H2DIDS but not NPPB, glibenclamide, or furosemide,
indicating that CFTR is not on the basolateral membrane and that an
unidentified cAMP-activated, DIDS-sensitive Cl
permeability is present on the basolateral membrane.
cAMP increases apical but not basolateral HCO
permeability.
Previous investigations have shown that CFTR is permeable to
HCO
(8, 35, 44). Because
HCO
transport is important for fluid transport in
BCEC, we examined whether CFTR could enhance HCO
permeability. A constant-CO2 protocol described previously
(4) was used to examine apical and basolateral
HCO
permeability of cultured corneal endothelial
cells. Perfusate [HCO
] concentration was reduced
from 28.5 mM at pH 7.5 (BR solution) to 2.85 mM at pH 6.5 (LB solution) while both apical and basolateral solutions were continually gassed with 5% CO2. The decreased [HCO
] and lower pH of the bath can both contribute to a drop in pHi.
H+ fluxes, however, were only 18% of the initial
dpHi/dt (4) and were unaffected by
FSK (data not shown). Figure 6 shows that there was a small drop in pHi when LB solution was
introduced on the apical side. After cells were returned to BR solution
perfusion on both sides, introduction of FSK caused a small but
rapid alkalinization followed by a new steady-state pHi
that was slightly below the baseline. In the continued presence of FSK
for 5 min, introduction of LB solution caused a faster and deeper
acidification than control. Figure 6B summarizes the results
from 14 experiments and shows that apical HCO
permeability was increased approximately twofold by FSK. After a 10-min
wash on the apical side with BR solution, the basolateral side was then
exposed to LB solution (Fig. 6A). Because of the presence of
the Na+-nHCO
cotransporter on the
basolateral side (45), there was a much greater
acidification than on the apical side. Cells were returned to BR
solution and FSK was then introduced for 5 min, followed by LB solution
on the basolateral side. The rate of acidification was
unaffected by FSK. Again, reversing the order of LB solution exposure
to basolateral followed by apical produced similar results. Figure
6B summarizes these results and indicates that FSK caused a
small but statistically insignificant decrease in
HCO
permeability on the basolateral side. Taken
together, these results indicate that apical, but not basolateral,
HCO
permeability is increased by cAMP.

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Fig. 6.
Effects of FSK on apical and basolateral
HCO fluxes. A: both apical and
basolateral compartments were initially perfused with
CO2-HCO Ringer (BR; 28.5 mM) solution.
Boxes indicate when BR was changed to low bicarbonate (LB; 2.8 mM)
solution on the apical or basolateral side. FSK (50 µM) was
preincubated 5 min in BR solution before the LB solution pulse. The
break in the data indicates a 10-min wash with BR solution on the
apical side. B: maximum change in intracellular pH
(pHi) per second. Values are means ± SE.
*Significantly different from the control (apical: n = 14, P < 0.05; basolateral: n = 5).
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To further confirm that FSK-activated apical HCO
permeability is caused by CFTR, the Cl
channel blockers
NPPB and glibenclamide were applied in the presence of FSK. Figure
7 shows the effect of 50 µM NPPB on
FSK-activated HCO
flux on the apical side. Because NPPB can partially absorb BCECF fluorescence excitation at 440 nm,
which induces an increase in F495/F440, paired
experiments were not possible. Therefore, we applied 50 µM NPPB
during the entire experiment. Figure 7 shows that FSK in the presence
of 50 µM NPPB only increased apical HCO
flux by
~38%. This represents a 59 ± 15% (n = 6, P < 0.05) inhibition of FSK-induced apical
HCO
flux by NPPB relative to FSK alone. Figure
8A shows the inhibition of FSK-induced HCO
flux on the apical side by
glibenclamide. After a 5-min preincubation with 50 µM FSK, LB
solution was introduced on the apical side, which caused a rapid
acidification of 1.6-fold greater than controls. After cells
were returned to BR solution on both sides, 100 µM glibenclamide + FSK was added to the apical perfusate and preincubated for 10 min. LB
solution was then introduced in the continued presence of FSK and
glibenclamide, which produced a 52% decrease in HCO
permeability relative to FSK alone. These results are summarized in
Fig. 8B and suggest that FSK-activated apical
HCO
permeability was caused, at least partially, by
the activation of apical CFTR.

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Fig. 7.
Effect of NPPB on FSK-induced apical
HCO flux. Both apical and basolateral compartments
were initially perfused with BR solution. NPPB (50 µM) was applied on
the apical side in the entire experiment. LB solution was first pulsed
on the apical side in the absence of FSK. After the reintroduction of
BR solution, 50 µM FSK was applied and preincubated for 5 min. LB
solution was then pulsed in the presence of FSK.
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Fig. 8.
Effect of Glib on FSK-induced apical
HCO flux. A: both apical and basolateral
compartments were initially perfused with BR solution. LB solution was
introduced on the apical side and repeated. After the reintroduction of
BR solution, 50 µM FSK was applied and preincubated for 5 min. LB
solution with FSK was then introduced. After a 10-min wash with BR
solution, cells were preincubated with 100 µM Glib together with 50 µM FSK for 5 min in BR solution followed by the LB solution pulse.
B: maximum change in pHi per second. Values are
means ± SE. #Significantly different from FSK alone
(n = 7, P < 0.05).
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Genistein increases apical Cl
and
HCO
permeability.
Recently, many reports have demonstrated that the isoflavone genistein
stimulates CFTR Cl
channel activity (2, 14, 18, 19,
28, 40, 49). Here, we investigated the effect of 50 µM
genistein on Cl
and HCO
fluxes in
BCEC. Figure 9 shows the effect of
genistein on apical Cl
permeability. Both sides were
initially perfused with Cl
-free Ringer solution. When
Cl
was added on the apical side, there was a small drop
in MEQ fluorescence that was strongly accelerated by the addition of
FSK. After a 10-min wash with Cl
-free Ringer solution on
the apical side, Cl
was added again to the apical side,
inducing a small drop in MEQ fluorescence. In the continued presence of
Cl
, addition of 50 µM genistein produced a sharp
decrease in MEQ fluorescence, consistent with genistein activating
CFTR. When genistein was combined with FSK and added to the apical
side, there was no further increase in Cl
permeability
over genistein alone. Figure 9B shows that 50 µM genistein
significantly increased apical Cl
permeability by
16-fold. Interestingly, Fig. 9B also shows that in these
paired experiments the genistein-induced increase in Cl
permeability was over fourfold stronger than that of FSK alone, which
is consistent with previous reports of genistein activation of CFTR
(40).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of genistein (Gen) on apical
Cl flux as measured by MEQ fluorescence
(F/F0). Both sides were initially perfused with
Cl -free (nitrate substituted) Ringer solution. Arrows
indicate the applications of Cl -rich Ringer solution, 50 µM FSK, and 50 µM Gen as well as the combination of FSK and Gen on
the apical side from the same Anodisc culture. B: maximum
%dF/F0 per minute. Values are means ± SE.
#Significantly different from FSK alone (n = 6, P < 0.05).
|
|
Figure 10 shows the effect of
genistein on apical HCO
flux under the
constant-CO2 protocol. Both sides were initially perfused
with BR solution. When the apical side was perfused with LB solution in
the presence of 50 µM genistein, acidification was significantly
faster relative to controls. In these same cells, LB solution perfusion
induced an acidification in the presence of 50 µM FSK that was
approximately the same as that with genistein. Figure 10A
also shows that, surprisingly, when 50 µM genistein was combined with
50 µM FSK, HCO
flux was reduced to control levels.
These results are summarized in Fig. 10B, which shows that
genistein alone can increase HCO
permeability to the
same extent as FSK but that there is a negative synergistic effect on
HCO
permeability by the two drugs.

View larger version (25K):
[in this window]
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|
Fig. 10.
Effect of Gen on apical HCO flux.
A: both apical and basolateral compartments were initially
perfused with BR solution. LB solution was then pulsed on the apical
side. After the reintroduction of BR solution, LB solution was pulsed
in the presence of 50 µM Gen. After a 10-min wash, cells were
preincubated with 50 µM FSK for 5 min before the LB solution pulse.
Finally, FSK was preincubated for 5 min, followed by 50 µM Gen in LB
solution. B: maximum change in pHi per second.
Values are means ± SE. #Significantly less than FSK or Gen alone
(n = 7, P < 0.05). C: same
experiment as A except in Cl -free (gluconate
substituted) Ringer solution and no preincubation with FSK just before
the last LB solution pulse with FSK and Gen combined. D:
maximum change in pHi per second. Values are means ± SE. #Significantly less than FSK or Gen alone (n = 9, P < 0.05)
|
|
Phosphorylation of CFTR can increase activation by flavonoids such as
genistein (18). However, at concentrations exceeding 50 µM flavonoids, prephosphorylation can be inhibitory. Considering the
possibility that the 5-min preexposure to FSK could sensitize CFTR to
being inhibited by genistein, the experiment was repeated except that
FSK, genistein, and LB solution were introduced simultaneously, (i.e.,
no preexposure to FSK, which is the same protocol as Cl
flux experiments shown in Figs. 3-5 and 9). The result, however, was the same; HCO
permeability in the combined
presence of FSK and genistein was reduced to control levels even though
there was no preexposure to FSK (n = 8, data not
shown). Finally, we considered the possibility that this negative drug
interaction on HCO
permeability may be influenced by
the presence of Cl
because Cl
permeability
is increased to such a large extent by genistein. To test this
possibility, the experiment was repeated in the absence of
Cl
(gluconate substituted). Figure 9C shows
that again when FSK and genistein were added together,
HCO
permeability was reduced to control levels.
Figure 9D summarizes these experiments. In the absence of
Cl
, genistein alone increased HCO
permeability by 1.67-fold, which is not significantly different than in
the presence of Cl
(1.77-fold; Fig. 9B). On
the other hand, in the absence of Cl
, FSK increased
HCO
permeability 4.2-fold compared with only
1.7-fold in the presence of Cl
. Presumably, this is due
to removal of a competitive inhibitor. Nevertheless, the absence of
Cl
had no effect on the inhibitory effect of the
FSK-genistein combination.
 |
DISCUSSION |
CFTR is expressed on the apical membrane of bovine corneal
endothelium.
Previously, we have shown by RT-PCR analysis that CFTR mRNA is present
in corneal endothelial cells (46). Immunoblotting (Fig. 1)
confirmed the expression of CFTR protein in both fresh and cultured
corneal endothelial cells. Indirect immunofluorescence confocal images
(Fig. 2) further confirmed the expression of CFTR and showed that CFTR
is at the same level as ZO-1 and not basolateral to ZO-1, indicating an
exclusively apical localization.
FSK-activated CFTR increases apical Cl
and
HCO
permeability.
Functional analysis from a wide variety of cell types has demonstrated
that CFTR can be activated by at least two different mechanisms. One is
a cAMP-dependent activation of protein kinase A, leading to the
phosphorylation of the R domain of CFTR and activation of the channel
(19, 24). Another is a cAMP-independent mechanism, which
is mediated by the addition of such channel openers as genistein (a
specific inhibitor of protein tyrosine kinases), which directly
interact with CFTR, activating and prolonging the open channel
conformation (14, 28, 40).
We examined FSK-activated Cl
permeability across apical
and basolateral membranes. Interestingly, FSK increased
Cl
permeability on both sides; however, the augmentation
on the basolateral side (~3-fold) is significantly lower than that on the apical side (~10-fold; Fig. 3). FSK-stimulated apical
Cl
fluxes were inhibited by NPPB and glibenclamide (Figs.
4 and 5), whereas H2DIDS had no effect, consistent with an
apically located CFTR. The negative effect of H2DIDS on
stimulated apical Cl
flux also suggests that the enhanced
Cl
flux is not via the DIDS-sensitive outwardly
rectifying Cl
channels, which can be secondarily
stimulated by activated CFTR (9, 21, 39). FSK-activated
basolateral Cl
permeability was unaffected by NPPB or
glibenclamide but significantly inhibited by H2DIDS,
consistent with CFTR not being present on the basolateral membrane.
FSK-activated basolateral Cl
permeability may be caused
by NKCC1 because it has been reported that NKCC1 can be activated by
cAMP (10, 13, 15, 27, 31, 41). Furosemide, however, had no
effect on the FSK-activated basolateral Cl
flux. This
result together with the H2DIDS sensitivity on the basolateral side indicates that NKCC1 is unlikely to be involved in
FSK-activated basolateral Cl
permeability. These data are
similar to those found in airway epithelium, which showed a basolateral
cAMP activated inwardly rectifying Cl
channel that is not
CFTR (48). Further studies are needed to fully
characterize this basolateral cAMP-dependent Cl
flux in
corneal endothelial cells.
Because corneal endothelial fluid transport is HCO
dependent and CFTR is permeable to both HCO
and
Cl
in a variety of cells (35, 42, 52), we
examined the effect of cAMP on apical and basolateral
HCO
permeability. We found that FSK-stimulated
HCO
flux was only present on the apical side and
that this was inhibited by NPPB and glibenclamide, consistent with the
HCO
flux being facilitated by CFTR. The increased
HCO
permeability shown here is not due to an anion
exchanger because anion exchange could not be demonstrated in cultured
corneal endothelium (7) and apical HCO
efflux is unchanged in Cl
-free, gluconate-substituted
solutions (4).
FSK did not enhance basolateral HCO
efflux on
exposure to LB solution, suggesting that the
Na+-2HCO
cotransporter, which is the major HCO
flux pathway on the basolateral side
(4), is not directly stimulated by cAMP. In fact, there was a small but not statistically significant decrease in basolateral HCO
efflux in the presence of FSK. This may be due
to the slightly lower baseline pHi caused by FSK (i.e.,
lower intracellular [HCO
] and slightly reduced
driving force for HCO
efflux). On the other hand, in
the presence of BR solution on both sides, FSK alone produced a
transient alkalinization quickly followed by acidification to below
resting pHi (see Fig. 6). The initial alkalinization may be
explained by the membrane potential (Em)
depolarization produced by FSK in BCEC (5), which would increase HCO
influx via basolateral Na+-nHCO
cotransport. We speculate that the initial burst of HCO
influx is then offset by
increasing apical HCO
efflux, leading to a new lower
steady-state pHi. These findings suggest that cAMP can
indirectly stimulate Na+-2HCO
via
Em depolarization from activation of apical CFTR
(and possibly basolateral cAMP-dependent channels), as suggested for
the pancreatic duct (43).
Genistein-stimulated Cl
and HCO
permeability.
Further evidence for the expression and functionality of CFTR in
corneal endothelial cells is the potent stimulation of Cl
and HCO
fluxes by genistein (Figs. 9 and 10).
Genistein (50 µM) alone produced a 16-fold increase in
Cl
permeability relative to control. This stimulation was
unaffected by addition of FSK, indicating that CFTR had been maximally
stimulated. Genistein alone also increased apical
HCO
permeability. However, most interestingly, the
combination of genistein and FSK (50 µM each) completely abolished
any increase in apical HCO
permeability. That this block occurred for HCO
and not Cl
permeability is puzzling. The protocols were slightly different in that
for Cl
flux FSK and genistein were added acutely, whereas
for HCO
flux cells were preincubated for 5 min with
FSK. Earlier studies (18) have shown that phosphorylated
CFTR is more easily activated by flavonoids, including genistein.
However, high concentrations of flavonoids (>50 µM) can inhibit
phosphorylated CFTR. Thus we tested whether the preincubation with FSK
may contribute to the inhibitory effect of genistein. Without
preincubation, the FSK-genistein combination still reduced
HCO
permeability to control levels (Fig. 10).
Finally, we considered the possibility that this negative drug
interaction on HCO
permeability may be influenced by
the presence of a competitive anion. Cl
flux experiments
were performed in the absence of HCO
, and there was
no inhibition by the FSK-genistein combination (Fig. 9). Therefore, we
repeated the HCO
flux experiments in the absence of
Cl
. Activation of HCO
permeability by genistein alone was unaffected; however, FSK-activated
HCO
permeability was significantly increased (Fig.
10), consistent with the removal of a competitive inhibitor.
Nevertheless, the FSK-genistein combination still reduced
HCO
permeability to control levels. Thus we conclude
that the FSK-genistein combination can inhibit CFTR, consistent with
earlier findings(18), but that the sensitivity to this
combination may be anion dependent.
Physiological implications.
Increased cAMP levels stimulate rabbit corneal endothelial fluid
secretion (12, 37). Thus an apical CFTR could play a significant role in stimulated ion-coupled fluid transport. In unstimulated cells, apical Cl
and HCO
permeability are approximately two and three times lower than
basolateral permeability, respectively (4, 20). Thus the
rate-limiting step in transendothelial anion transport is at the apical
membrane. Basolateral Cl
and HCO
permeability are predominantly due to the
Na+-K+-2Cl
cotransporter and the
Na+-HCO
cotransporter (45,
46), respectively. Here, we show that FSK stimulation increases
both apical and basolateral Cl
permeability so that
apical is now twice basolateral permeability. FSK stimulation also
increases apical HCO
permeability but not
basolateral, so that apical and basolateral permeability become
essentially equal. These results suggest that cAMP enhanced fluid
transport in corneal endothelium could be due directly or indirectly to
activation of CFTR.
The relative contributions of Cl
and
HCO
fluxes to baseline (i.e., unstimulated)
endothelial fluid transport are not precisely known. Tracer flux
experiments and electrophysiology are hampered by the extreme leakiness
of the fresh or cultured corneal endothelial preparation
(R = 25
· cm
2,
transendothelial potential =
0.5 mV). Nevertheless, we do
know that both Cl
(50) and
HCO
(11, 17, 23, 36) are needed to
maintain corneal hydration. Removal of HCO
, application of DIDS, or carbonic anhydrase inhibitors (36)
prevent dehydration of edematous corneas, whereas in the presence of
bumetanide corneal hydration can return to normal (38,
50). Previously, it has been shown that bumetanide has no effect
on normally hydrated corneas (38); however, this conflicts
with a recent report indicating that bumetanide can cause a small
amount of swelling (~6%) of normally hydrated corneas
(22). Furthermore, furosemide has no effect on
intracellular [Cl
] (20) unless cells are
stimulated by cAMP (5). On balance, these findings do not
favor a strong role for
Na+-K+-2Cl
cotransport; however,
they do not exclude a direct contribution of Cl
flux to
corneal endothelial fluid transport. Cl
fluxes could also
contribute indirectly to supporting HCO
transport.
For example, conductive efflux of Cl
, either in the
resting or cAMP stimulated condition, will depolarize Em, thereby dissipating the hyperpolarizing
effects of the basolateral Na+-2HCO
cotransporter and thus promoting continuous uptake of
HCO
. In fact, we have shown that
HCO
flux through the
Na+-2HCO
cotransporter is slowed in
gluconate-substituted Cl
-free solutions (4).
At the apical membrane, either anion has the potential to contribute to
net transendothelial flux; however, the greater sensitivity of fluid
transport to HCO
transport inhibitors favors a role
for HCO
over Cl
. Interestingly,
bicarbonate-activated adenylyl cyclase, which is not activated by FSK,
can increase [cAMP] in BCEC by 56% compared with that in the absence
of HCO
(33). Thus it is conceivable
that CFTR has a role in baseline fluid transport as well.
In general, CF patients do not have visual or ocular problems. Corneal
transparency and function appear to be normal. One study
(25) has shown that CF patients have slightly thicker corneas, ~4% greater than normal. This may represent a small level of edema, but it is not enough to effect corneal transparency. Interestingly, there are corneal endothelial morphological differences between normal and CF patients. CF patients have a significantly higher
cell density (18%) than normal patients (25). This may be
a sign of compensation for the CFTR defect. For the same surface area
of the cornea, an endothelial layer with higher cell density will have
greater lateral membrane surface area. Thus the lateral membrane may
have a greater concentration of Na+-K+-ATPase
or Na+-2HCO
cotransporters. It is not
clear how this could compensate for an apical anion channel defect, but
it indicates that the endothelial cells have responded to the defect.
To determine whether the morphological change is related to transport
function will require further investigation.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institutes of Health Grant
EY-08834.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
J. A. Bonanno, Indiana Univ. School of Optometry, 800 E. Atwater Ave., Bloomington, IN 47405 (E-mail:
jbonanno{at}indiana.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00384.2001
Received 9 August 2001; accepted in final form 25 November 2001.
 |
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