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Indiana University School of Optometry, Bloomington, Indiana 47405
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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HCO
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 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 and HCO pathway on the basolateral
membrane that is not CFTR.
cornea; endothelium; chloride permeability; MEQ; bicarbonate permeability; intracellular pH; BCECF; forskolin; cAMP; genistein
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 (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 are loaded
into corneal endothelial cells, to levels above electrochemical equilibrium (5), on the basolateral (stromal) side by the
Na+-2HCO cotransporter (NKCC1)
(20, 22), respectively. What is less clear, however, is
the mechanism for apical anion efflux.
Three possible mechanisms for HCO/HCO 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
(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
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 protein1 · ml IP
buffer1) 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
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 s1) 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 HCO1, and ratios were
calibrated against intracellular pH (pHi) by the high
K+-nigericin technique (47). Anodiscs were
perfused on both sides with HCO
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
, 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 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.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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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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% min1). 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% min1) 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% min1) 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% min1) 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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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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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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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
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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
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Genistein increases apical Cl and
HCO channel activity (2, 14, 18, 19,
28, 40, 49). Here, we investigated the effect of 50 µM
genistein on Cl and HCO 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).
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flux experiments shown in Figs. 3-5 and 9). The result, however, was the same; HCO 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, genistein alone increased HCO (1.77-fold; Fig. 9B). On
the other hand, in the absence of Cl, FSK increased
HCO. 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.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 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 in a variety of cells (35, 42, 52), we
examined the effect of cAMP on apical and basolateral
HCO-free, gluconate-substituted
solutions (4).
FSK did not enhance basolateral HCOGenistein-stimulated Cl and HCO
and HCO 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 is puzzling. The protocols were slightly different in that
for Cl flux FSK and genistein were added acutely, whereas
for HCO flux experiments
were performed in the absence of HCO. Activation of HCO
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 and HCO cotransporter and the
Na+-HCO permeability so that
apical is now twice basolateral permeability. FSK stimulation also
increases apical HCO
and
HCO
· cm2,
transendothelial potential = 
0.5 mV). Nevertheless, we do
know that both Cl (50) and
HCO] (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, either in the
resting or cAMP stimulated condition, will depolarize Em, thereby dissipating the hyperpolarizing
effects of the basolateral Na+-2HCO-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. 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| |
ACKNOWLEDGEMENTS |
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This study was supported by National Institutes of Health Grant EY-08834.
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FOOTNOTES |
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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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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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