Oxidant and antioxidant modulation of chloride channels
expressed in human retinal pigment epithelium
T. X.
Weng1,
B. F.
Godley2,3,
G. F.
Jin4,
N. J.
Mangini5,
B. G.
Kennedy6,
A. S. L.
Yu7, and
N. K.
Wills1,4
1 Department of Physiology and Biophysics,
University of Texas Medical Branch, Galveston 77555;
2 The Retina Foundation of the Southwest, Dallas
75231; 3 Department of Ophthalmology, University of
Texas Southwestern, Dallas 75231; 4 Department of
Ophthalmology and Visual Sciences, University of Texas Medical Branch,
Galveston, Texas 77555; Departments of 5 Anatomy and
6 Physiology, Indiana University School of Medicine,
Gary, Indiana 46408; and 7 Renal Division, Brigham
and Women's Hospital, Harvard Institutes of Medicine, Boston,
Massachusetts 02115
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ABSTRACT |
Retinal pigment epithelium (RPE)
possesses regulated chloride channels that are crucial for
transepithelial fluid and ion transport. At present, little is known
about the molecular nature of chloride channels in human adult RPE
(haRPE) or the effects of oxidative stress on membrane conductance
properties. In the present study, we assessed ClC channel and cystic
fibrosis transmembrane conductance regulator (CFTR) expression and
membrane chloride conductance properties in haRPE cells. ClC-5, ClC-3,
ClC-2, and CFTR mRNA expression was confirmed with RT-PCR analysis, and
protein expression was detected with Western blot analysis and
immunofluorescence microscopy. Whole cell recordings of primary
cultures of haRPE showed an outwardly rectifying chloride current that
was inhibited by the oxidant H2O2. The
inhibitory effects of H2O2 were reduced in
cultured human RPE cells that were incubated with precursors of
glutathione synthesis or that were stably transfected to overexpress glutathione S-transferase. These findings indicate a
possible role for ClC channels in haRPE cells and suggest possible
redox modulation of human RPE chloride conductances.
immunocytochemistry; patch clamp; glutathione; glutathione
S-transferase
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INTRODUCTION |
RETINAL PIGMENT
EPITHELIUM (RPE) comprises part of the
blood-retina barrier and functions in several processes that
are vital for the preservation of sight. Among the crucial roles of
this epithelium is the regulation of the volume and electrolyte
composition of the subretinal space. This function is achieved largely
by regulated transepithelial transport of chloride from the
subretinal space to the choroid with the obligatory movement of
water. Disruption of RPE chloride transport can result in the
accumulation of fluid in the subretinal space and subsequent retinal
detachment (3, 26).
Chloride transport across the RPE is mediated in part by chloride
channels that are stimulated by calcium and cAMP (Ref. 28; cf. Refs. 13, 14). In recent whole cell
patch-clamp recordings in SV40-transformed cultured human fetal RPE
(hfRPE) cells, we (39) identified an outwardly rectifying
chloride current that was stimulated by cAMP but was inhibited by the
chloride channel blocker DIDS, acidic bathing solutions, or low
concentrations of the oxidative agent H2O2. The
molecular identity of the chloride channel(s) responsible for this
current was not determined. However, these cells expressed several
candidate chloride channels including cystic fibrosis transmembrane
conductance receptor (CFTR) and members of the ClC chloride channel
family (ClC-2, ClC-3, and ClC-5; Ref. 39). At present,
little is known about the expression of ClC chloride channels in the
intact human RPE.
Recent evidence suggests that the ClC family of voltage-gated chloride
channels may be crucial for retinal function. At least three members of
this family are associated with retinal degenerations in mice.
Specifically, transgenic mice that were deficient for ClC-3
(34), ClC-2 (6), or ClC-7 (17)
were found to develop retinal degenerations and blindness within weeks
after birth. The basis of these degenerations is not presently understood.
The finding of ClC channel expression in human fetal cells and the
consequences of their deletion in mice indicate a possible functional
role for these channels in the developing human RPE. To date, there
have been no studies of ClC channel expression in the intact human
adult RPE (haRPE). In addition, it is unclear whether regulation of RPE
cell membrane conductances is similar in human fetal and adult cells.
In the present study, RT-PCR and immunocytochemical analysis methods
were used to assess CFTR and ClC channel expression in haRPE cells. In
addition, we determined whether an oxidant-regulated chloride
conductance was present in primary cultures of haRPE cells and compared
this conductance with those previously assessed in cultured hfRPE
cells. Finally, we assessed whether antioxidants can protect RPE cell
chloride conductances from the inhibitory effects of oxidative agents.
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METHODS |
Immunocytochemistry.
Post mortem eyes were obtained from the National Disease Research
Interchange under local Institutional Review Board approval for the use
of human tissue. For the present immunocytochemistry study, a
donor eye from a 66-year-old Caucasian man was fixed in 4%
formaldehyde in phosphate-buffered saline at ~11 h post mortem.
RPE-choroid patches were dissected, and 6-µm-thick sections were
obtained by cryostat. Before antibody incubation, sections were
permeabilized with 1% SDS and then treated with 0.025% potassium permanganate (cf. Ref. 16) to reduce the significant
autofluorescence of lipofuscin and Bruch's membrane that is
characteristic of native human RPE tissue (10). Sections
were incubated in commercially available polyclonal primary antibody
raised against rat ClC-3 (Alomone Labs, Jerusalem, Israel), ClC-5 (C1
antibody raised against residues 570-677 of rat ClC-5; Ref.
21) or CFTR monoclonal antibody (formerly Genzyme; now
MAB25031, R&D Systems, Minneapolis, MN). The secondary fluorescent
antibodies were Alexa 488-conjugated goat anti-rabbit IgG or goat
anti-mouse IgG (Molecular Probes). Sections were mounted in Vectashield
containing DAPI (Vector Labs) to stain cell nuclei (blue emission).
Antibody localization was visualized with an inverted microscope
equipped for epifluorescence (Zeiss Axiovert 100M) and analyzed with
deconvolution data analysis software (Slidebook; Intelligent Imaging
Innovations, Denver, CO). To distinguish residual autofluorescence from
specific immunoreactivity, images were obtained with sequential
excitation with narrow-bandwidth fluorescence filters for FITC (green
emission) and CY3 (red emission). The autofluorescent materials have
broad spectral properties (24) and were visible with both
filters, whereas the Alexa-488 fluorescence was visible only with the
FITC excitation filter. By this method, autofluorescent materials
appeared yellow or red-orange whereas Alexa-488-labeled components
appeared green. Digital images were further processed with Photoshop
5.5 (Adobe Systems, Seattle, WA).
Primary RPE cell culture (haRPE).
Human donor eyes were obtained from the Oregon Eye Bank within 24 h of death and stored in a moist chamber at 4°C. The RPE was removed
under sterile conditions and dissociated by treatment with EDTA at
37°C for ~20 min, and the suspended cells were then subjected to
centrifugation (3,000 g) in growth medium consisting of DMEM
containing 20% fetal bovine serum. Cells were plated on plastic or
poly-D-lysine-coated glass coverslips at a subconfluent density and placed in a humidified incubator maintained at 37°C with
an atmosphere of 95% air-5% CO2. Cells were fed two or
three times a week with Eagle's minimum essential medium (MEM; Sigma Aldrich, St. Louis, MO) supplemented with 1% penicillin and
streptomycin and 10% fetal bovine serum (FBS; Hyclone Laboratories,
Logan, UT). The cells were passaged between two and four times and
grown for 2-5 days before whole cell patch-clamp investigations.
Cultured hfRPE cells.
SV40-transformed hfRPE cells (RPE 28 SV4; Coriell Institute, Camden,
NJ) were grown in MEM (Sigma-Aldrich) supplemented with 1% penicillin
and streptomycin and 10% FBS (Hyclone Laboratories) as previously
described (39). The cells were plated at subconfluent density on glass coverslips coated with poly-D-lysine. The
cells were incubated in a humidified incubator at 37°C in 95% air
and 5% CO2 overnight or were grown to confluence for a
period of 3-7 days.
Stably transfected cells that overexpress human glutathione
S-transferase (GST) A1.1 and control cells stably
transfected with expression vector alone were produced by exposing
80-90% confluent hfRPE cells to plasmid DNA and Transfast
transfection reagent (Promega, Madison, WI) in DMEM at 37°C in a
CO2 incubator for 1 h. Cells were subsequently
overlaid with 4 ml of 10% FBS in DMEM and incubated another 48 h.
Positive cells were then selected by using G418 antibiotic (600 µg/ml) for a period of 2 wk. The culture medium was replaced three
times per week. Individual clones were trypsinized, and single cells
were transferred to a 96-well plate. Colonies grown from single cells
were transferred to flasks and grown to confluence for subsequent use.
In experiments investigating the effects of glutathione precursors,
nontransfected SV40-transformed hfRPE cells were incubated in fresh
culture medium containing (in mM) 0.5 glutamate, 0.5 glycine, and 0.1 cysteine for 1 h before whole cell current measurements.
Whole cell patch-clamp recording.
Membrane ionic currents were recorded with conventional tight-seal
whole cell patch-clamp techniques (12) under conditions of
symmetrical chloride concentrations in the absence of potassium ions.
The composition of the solutions were as follows (in mM): bath, 130 tetramethylammonium chloride (TMA-Cl), 2 NaH2PO4, 2 calcium cyclamate, 1 MgSO4, 5 glucose, and 10 HEPES; pipette, 130 TMA-Cl, 0.2 calcium cyclamate, 3 MgSO4, 2 EGTA, 10 HEPES, and 3 Na-ATP.
The pH of the solutions was 7.4, and the osmolalities were 300 and 270 mosmol/kgH2O, respectively. Borosilicate glass (cat. no. 18150F-3; WPI, Sarasota, FL) pipettes were pulled (model P-87; Sutter Instrument, Novato, CA) and fire-polished to a tip diameter of 1-2 µm (tip resistance = 3-6 M
). The
pipette was connected to the head stage of a patch-clamp amplifier
(Axopatch 200B; Axon Instruments, Foster City, CA), and the bath was
connected to ground with a 3 M KCl agar bridge. For recordings, the
pipette was positioned next to the cell with a low-drift
micromanipulator (Burleigh PCS-5200) under observation with an inverted
phase-contrast microscope (Zeiss model IM). Membrane voltages were
corrected for liquid junction potentials.
Stimulus control and data acquisition and processing were carried out
with a Pentium PC and analog-digital interface, using commercially
available data acquisition and analysis software (DigiData 1200 and
pCLAMP 6.03 software; Axon Instruments). Electrode offset was balanced
before forming a gigaseal. The electrode capacitance and series
resistance were compensated with the amplifier's analog circuitry.
Solution junction potentials were negligible (<3 mV). Currents were
low-pass Bessel-filtered at 5 kHz and digitized at 10 kHz for storage
and analysis. Solution changes and drug delivery were achieved by a
gravity drive superfusion system.
Drugs and antibodies.
Glutathione, H2O2, glutamate, glycine and
cysteine were obtained from Sigma-Aldrich. Unless otherwise noted,
polyclonal antibody for ClC-3 was from Alomone Labs and for ClC-5 was
antiserum C1 raised against the COOH-terminal region of rat ClC-5
(21). Monoclonal CFTR antibody was from Genzyme (cat
no.2503-01, now MAB25031, R&D Systems).
Western blot analysis.
Human RPE was first homogenized then lysed in nonreducing Laemmli
sample buffer following the methods of Marmorstein et al. (23). Cell lysates were electrophoresed on a precast 10%
SDS-polyacrylamide gel (pager Gold; Cambrex, Rutherford, NJ) and
transferred onto a polyvinylidene fluoride membrane (Millipore,
Bedford, MA). Alkaline-phosphatase-conjugated secondary antibodies and
nitro blue tetrazolium/5-bromo-4-chloro-3-indoylyl phosphate (Western
Blue Stabilized Substrate; Promega) were used for signal detection.
RT-PCR.
Total RNA was isolated from primary cultures of human adult RPE cells
with TRIzol (GIBCO) following the methods of Mangini et al.
(22). For single-strand cDNA synthesis, 5 µg of total RNA from each sample was reverse transcribed with Superscript II
reverse transcriptase (GIBCO, Gaithersburg MD) and oligo-dT priming.
Amplification was performed with 2-µl aliquots of cDNA, High-Fidelity
PCR Master (Roche Diagnostics, Indianapolis, IN), and specific primer
sets for ClC-2, ClC-3, ClC-5, and CFTR. Primer sets were based on
published sequences that spanned at least one intron-exon junction as
follows: human ClC-2 primer set (accession no. NM004366) designed to
amplify a segment from 104 to 515 (sense, 5'-aagaggaagctgctcggatt-3';
antisense, 5'-cgcaagatggtcttcatctc-3'); human ClC-3 primer set
(accession no. NM001829) to amplify a segment from 375 to 1205 [sense
5'untranslated region (UTR), 5'-gctccgagggtagctaggtt-3'; antisense
ClC-3, 5'-agagccacaggcatatggag-3']; human ClC-5 primer set (Ref. 11;
accession no. X91906) to amplify segment 686 to 1006 (sense,
5'-gagcctttgcctacatagtcaat-3'; antisense ClC-5 5'-gcttggcttcattcttcctgta-3'). Two CFTR primer sets (accession no. XM004980) were used. The CFTR1 primer set was designed to amplify a
segment from 50 to 499 (sense, gcaggcacccagagtagtaggtc; antisense,
gataaatcgcgatagagcgttcct). The CFTR2 primer set was designed to amplify
a segment from 2467 to 3170 (sense, caaggtcagaacattcaccg; antisense,
gttgtaaaactgcgacaact). The expected lengths of the PCR products for
ClC-2, ClC-3, ClC-5, CFTR1, and CFTR2 are 411, 830, 320, 449, and 703 bp, respectively. PCR reactions were carried out in a GeneAmp 2700 thermocycler (Applied Biosystems, Foster City, CA) under the following
conditions: initial denaturation at 94°C for 3 min; 30 cycles of
94°C 30 s, 60°C 30 s, 72°C 1 min, and then 72°C 10 min (final extension). PCR products (5 µl) were electrophoresed with
2% agarose-1× TAE gels containing 0.5 µg/ml ethidium bromide. PCR
DNAs were purified for direct sequencing with the Wizard PCR DNA
purification system (Promega).
Data analysis and statistics.
Steady-state currents were used to calculate the current-voltage
(I-V) relationships. To calculate mean ± SE values,
the raw data (contained in Axon binary files or and Axon XYM files)
were exported to Excel spreadsheets. Paired t-tests or
nonparametric tests were used to evaluate statistical significance, as
appropriate (Instat; GraphPad Software, San Diego, CA). Graphs and
further analysis were generated with SigmaPlot5 software (Aurora, CO).
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RESULTS |
Rationale for choice of experimental preparations.
The following experiments used three different human RPE preparations.
For haRPE, intact native epithelium was used to assess ClC chloride
channel expression in Western blot analysis and in immunofluorescence
microscopy experiments. Primary cultures of these cells were used to
achieve whole cell patch-clamp recordings and for RT-PCR analysis to
minimize contamination from other subretinal cell types. Finally, a
continuous hfRPE cell line (39) was used to enable genetic
or environmental manipulation of antioxidative agents.
PCR and Western blot analysis.
To obtain evidence for the presence of ClC and CFTR gene products in
haRPE cells, RT-PCR analysis was performed with mRNA obtained from
primary cultures of haRPE cells. As shown in Fig. 1, RT-PCR performed with specific primer
sets for human ClC-2, ClC-3, and ClC-5 resulted in amplification of
fragments of the predicted size and sequence for these channels.
Similar experiments with CFTR-specific primers sets resulted in the
amplification of cDNAs with the appropriate predicted sizes and
sequences for CFTR (see Fig. 2). In
addition, as shown in Fig. 3, Western
blots of membrane proteins from intact haRPE (a gift of Dr. Alan
Marmorstein) probed with antibodies raised against the NH2
terminus of ClC-5 (a gift of Dr. O. Devuyst; see Ref. 36)
and ClC-3 (antibody 3A4, a gift of Dr. Thomas Jentsch; see Ref.
34) resulted in stained bands of the expected sizes for
ClC-3 and ClC-5.

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Fig. 1.
Agarose gel electrophoresis of PCR products amplified
with specific primer sets for ClC-2, ClC-3, and ClC-5 using cDNA
prepared from 2 independent primary cultures of human adult (ha)
retinal pigment epithelium (RPE) (Ex and Ya).
Products of the expected size were obtained with pairs of sense and
antisense primers. For details of primer sequences, see
METHODS.
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Fig. 2.
Agarose gel electrophoresis of PCR products amplified
from primary culture of haRPE (Ya) with 2 different cystic
fibrosis transmembrane conductance receptor (CFTR)-specific primer sets
(CFTR1 and CFTR2; see METHODS). Products of the expected
size and sequence were obtained with both sets of primers.
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Fig. 3.
Western blot of human RPE membranes probed with
antibodies against ClC-5 (lane 1) and ClC-3 (lane
2). Each lane is from a separate blot of the same protein
sample.
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Immunocytochemistry studies.
Figures 4-6 present images of cross-sections of native
haRPE-choroid, oriented with the RPE cell layer at the top and the
choroid below. Figure 4 presents a
typical example of a differential interference contrast image. Bruch's
membrane is indicated by the triangle and the RPE apical microvillus
membrane is at the top of the section. Figure
5 shows fluorescence images of two
adjacent sections from the same eye that were processed using a
deconvolution algorithm. The inverted white triangles indicate the
apical surface of the RPE cells. (Cell nuclei were stained with DAPI
and appear blue. Autofluorescence of the RPE cells and Bruch's
membrane appears red-orange.) In Fig. 5A, the cells were
stained with primary antibody for ClC-5. Staining was most prominent in
the RPE near the apical membrane. Some faint staining is also evident
in the choroid and capillary endothelial cells. In Fig. 5B,
the section was treated identically except that primary antibody was
incubated with ClC-5 peptide before incubation with the tissue. Note
that a marked reduction of specific ClC-5 staining occurred.

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Fig. 4.
Typical differential interference contrast image of a
cross-section of haRPE-choroid. From top to
bottom are shown the RPE layer, Bruch's membrane
(arrowhead), and underlying choroid. Scale bar = 22 µm.
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Fig. 5.
ClC-5 staining of haRPE-choroid. A: deconvolution
pseudo-color fluorescence image of a cross-section of haRPE. The apical
membrane is indicated by the arrowhead. Cells were treated with primary
antibody for ClC-5 and secondary Alexa-488 fluorescent antibody
(green). Staining was most prominent in the RPE apical membrane region,
with some staining in the choroid. B: similar image from an
adjacent section from the same donor. The apical membrane is indicated
by the arrowhead. Incubation was identical except for the presence of
antigenic blocking peptide. ClC-5 staining was markedly reduced. Cell
nuclei were stained with DAPI and appear blue in A and
B. Autofluorescence of the RPE cells and Bruch's membrane
appears orange-red. Scale bars = 22 µm.
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Fig. 6.
Deconvolution pseudo-color fluorescence images of native
haRPE-choroid (see Fig. 2). A: cells were stained with
primary antibody for ClC-3 and Alexa 488-labeled secondary antibody
(green). As in the case of ClC-5, staining is present in the RPE apical
membrane (arrowhead) and in the underlying choroid. Note the lack of
reactivity along the basolateral membrane. ClC-3 staining was
eliminated by incubation with antigenic peptide (data not shown).
B: section stained with a monoclonal antibody against CFTR.
Staining is present along the RPE apical membrane (top
arrowhead) and is also detectable in the basolateral membrane
(bottom arrowhead).
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Figure 6A shows the results
for a similar experiment using an antibody for ClC-3. As in the case of
ClC-5 antibody, strong positive ClC-3 antibody staining was detected
along the apical membrane. Preincubation of ClC-3 antibody with
antigenic peptide blocked the staining (data not shown). Figure
6B illustrates tissue stained with an antibody against CFTR.
Staining again was prominent along the RPE apical membrane but was also
detectable along the lateral cell margins and on the basal RPE surface
(Fig. 6B).
Whole cell membrane properties.
To assess membrane chloride conductances, whole cell currents were
recorded in primary cultures of haRPE cells using symmetrical chloride
solutions with the absence of potassium and low sodium (see
METHODS). Currents were stable within 2 min of obtaining a
whole-cell configuration. Figure 7
compares membrane chloride current measurements recorded from primary
cultured haRPE cells and SV40 transformed hfRPE cells
(39). Figure 7, A and B, presents representative current recordings from haRPE and hfRPE cells, respectively, showing the response to voltage steps ranging from
100
to +100 mV in 20-mV increments. In both cases, the currents showed
moderate outward rectification and little time dependence. The outward
rectification is also evident in Fig. 7C, which presents average I-V relationships calculated from steady-state
current values for both cell types (haRPE cells, n = 11; hfRPE cells, n = 8).

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Fig. 7.
Whole cell currents recorded in primary cultured haRPE cells
and SV40-transformed human fetal RPE (hfRPE) cells under conditions of
symmetrical chloride concentrations. A and
B: representative current responses from haRPE and hfRPE
cells over a holding potential range of 100 to +100 mV (20-mV voltage
step increments). C: comparison of mean steady-state
current-voltage relationships ( , haRPE cells;
, hfRPE cells).
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Figure 8 summarizes the effects of
replacing chloride in the bathing solution with impermeant anions. As a
first step in these experiments, TMA-Cl in the bathing solution was
replaced by NaCl (Fig. 8). No significant changes were noted in the
currents. In contrast, substitution of chloride by 130 mM cyclamate or
iodide led to decreases in the membrane currents. Permeability ratios calculated with a modified version of the Goldman-Hodgkin-Katz equation
(39) yielded a relative anion permeability sequence of
1:0.66 ± 0.21:0.55 ± 0.18 for chloride:iodide:cyclamate,
respectively (n = 4).

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Fig. 8.
Effects of anion replacement on whole cell currents in
primary cultures of haRPE cells. Outward currents were reduced when
chloride ( ) in the bathing solution was replaced by
iodide ( ) or by cyclamate ( )
(n = 4). Note that no difference in the currents was
apparent between the control solution [tetramethylammonium (TMA),
] and the NaCl solution.
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To further assess the ionic nature of the current, the chloride channel
blockers chlorotoxin (1 µM) and DIDS (1 mM) were each applied to the
bath. As shown in Fig. 9A,
addition of chlorotoxin reduced the whole cell current by 34.5 ± 7% (at +100 mV) and the conductance by 46 ± 13%
(n = 4) within 2 min (i.e., time required for complete
change of the bath solution). As shown in Fig. 9B, addition
of DIDS had a similar effect, reducing the conductance and current by
43.6 ± 11% and 37.2 ± 7%, respectively (n = 4).

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Fig. 9.
Effects of chloride channel blockers on membrane currents in
haRPE cells. A: addition of chlorotoxin (1 µM) to the
bathing solution significantly inhibited the currents. B:
DIDS addition (1 mM) also significantly reduced the currents (for
details, see text).
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Membrane conductance regulation.
To test for possible modulation of the membrane chloride conductances
in the primary cultured haRPE cells, several agents were used. Because
CFTR is activated by cAMP and chloride conductances in hfRPE cells and
RPE cells from other species have been shown to be increased by this
agent, we assessed the effects of a cAMP cocktail that contained 250 µM 8-bromo-cAMP (8-BrcAMP), 100 µM IBMX, and 25 µM forskolin. As
shown in Fig. 10, the current was increased by 39 ± 9% and the conductance was increased by
43 ± 17% mV (n = 4) within 3 min of the
application of the cAMP cocktail.

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Fig. 10.
Effects of a cAMP cocktail (250 µM cAMP, 100 µM
IBMX, and 25 µM forskolin) on whole cell currents recorded from
primary cultures of haRPE cells (n = 4).
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In contrast to the effects of cAMP, acute exposure to
H2O2 inhibited the chloride conductance of
SV40-transformed hfRPE cells (39). As shown in Fig.
11, a similar inhibitory effect of
H2O2 was observed for the chloride conductance
of primary cultures of haRPE cells. Figure 11A shows the
mean steady-state I-V relationships for these experiments in
the absence of H2O2 and after addition of 100 µM H2O2 to the bathing solution. Outward
currents (at +100 mV) were reduced by 38% ± 11%, and the slope
conductance at positive potentials (between +60 and +120mV) summarized
in Fig. 11B was reduced by 37 ± 9% (n = 7). Currents and slope conductance at negative potentials were also
significantly decreased by 35% ± 10% for currents at
60 mV and by
32% ± 11% for the slope conductance between
60 and
100 mV. The
inhibitory effect typically occurred within 1 min of exposure and
usually could be reversed by washing the cells with bath solution.

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Fig. 11.
Effects of addition of 100 µM H2O2 in
primary cultures of haRPE cells (n = 7). A:
mean steady-state current-voltage relationship before
( ) and after ( ) addition of
H2O2 (100 µM). B: whole cell
conductance data for the same experiments.
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Effects of antioxidative agents.
Glutathione, or GSH, is a naturally occurring substance that protects
cells, tissues, and organs from toxic free radicals and disease and has
been implicated in the modulation of the activity of several enzymes
(2,32). Glutathione is a tripeptide consisting of three
amino acids, glycine, glutamate, and cysteine, and each of these three
precursors is necessary for the manufacture of glutathione within
cells. Most intracellular glutathione is normally in the reduced state
(GSH), where it plays a protective role by scavenging free radicals or
by serving as a substrate for conjugation by enzymes that detoxify
harmful substances such as lipid peroxides.
In preliminary experiments, we sought to test whether exogenous
application of extracellular glutathione abolished the inhibitory effects of H2O2 on membrane currents. Although
we initially observed that glutathione abolished the inhibitory effects
of H2O2 on the membrane chloride conductance,
this experiment proved to be confounded. Measurements of
H2O2 concentrations revealed that glutathione addition directly decreased the quantity of
H2O2 in the extracellular bathing solution.
Therefore, we decided to test the effects of antioxidants by altering
intracellular glutathione levels or by genetically manipulating the
expression levels of antioxidant enzymes. Because a continuous RPE cell
line was needed for the genetic manipulation experiments, these studies
were carried out on the cultured hfRPE cell line used in our previous
study (39).
To alter intracellular glutathione levels, cells were exposed to
precursors of glutathione production (in mM: 0.5 glutamate, 0.5 glycine, and 0.1 cysteine) for 1 h before the experiments. As
previously shown by Sternberg and coworkers (7, 33), these supplements significantly increase glutathione production and protect
against oxidant-induced cell death. As shown in Fig.
12A, when the
SV40-transformed hfRPE cells were previously exposed to culture medium
supplemented with glutathione synthesis precursors, membrane currents
were not significantly affected by subsequent H2O2 exposure [
I =
11 ± 4% at +100 mV; not significant (n.s.); n = 4]. However, paired cells from the same batch that were bathed in
normal culture medium (without supplementation) did show significantly decreased currents (
I = 38 ± 8%; n = 3) in agreement with results from our previous study
(39). Figure 12B presents conductance data from the supplement-treated cells summarized in Fig.
12A. As in the case of the whole cell currents, the membrane
conductance in the treated cells was not significantly altered by
H2O2 treatment (12% ± 4%; n.s.). In
contrast, the conductance decreased by 45 ± 11%
(P < 0.05; t-test = 2.86) in cells
that were not exposed to supplements.

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Fig. 12.
Effects of H2O2 addition on
SV40-transformed hfRPE cells grown in culture medium supplemented with
glutathione precursors (n = 4). A: mean
steady-state current-voltage relationship before ( ) and
after ( ) addition of H2O2 (100 µM). B: whole cell conductance data for the same
experiments.
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Intracellular GSH is necessary for the detoxification of
oxidant-induced toxins by the enzyme GST. To test the possible role of
this antioxidant enzyme in protecting against the effects of oxidants,
SV40-transformed hfRPE cells that were stably transfected with cDNA
encoding human GST A1.1 were used. Godley and co-workers (unpublished
observations) have demonstrated that GST activity is increased twofold
in these cells. Figure 13 compares the
effects of 100 µM H2O2 on the membrane
chloride conductance in SV40-transformed hfRPE cells that were stably
transfected with vector alone (control) or with vector plus GST cDNA.
As shown in Fig. 13A, currents in control cells were reduced
by 36 ± 9% (n = 4), similar to the effects in
both primary cultured haRPE cells (Fig. 11) and nontransfected SV40-transformed hfRPE cells (39). Figure 13B
shows the results of identical experiments performed on
SV40-transformed hfRPE cells that overexpress GST A1.1. The inhibitory
effect of H2O2 on the membrane current was
abolished (
I =
4 ± 2%; n.s.; n = 4). The effect of H2O2 on the membrane
conductance was also abolished as summarized in Fig. 13C.
The change in conductance for the GST-transfected RPE cells was
3 ± 1% (n.s.; n = 4) compared with a decrease
of
38 ± 12% for the vector-transfected RPE cells
(P < 0.05; n = 4).

View larger version (17K):
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|
Fig. 13.
Effects of H2O2 addition on
SV40-transformed hfRPE cells stably transfected with vector alone
(A; n = 4) or vector containing cDNA
encoding glutathione S-transferase (GST, B;
n = 4). C: conductance data from the same
cells summarizing values before and after addition of
H2O2.
|
|
 |
DISCUSSION |
The results of these experiments provide new evidence for ClC
channel expression in the intact haRPE. In addition, an outward chloride conductance was detected in primary cultures of these cells
that was stimulated by cAMP and inhibited by
H2O2. These data confirm and extend our
previous results for cultured SV40-transformed hfRPE cells
(39). In addition, the present results provide the first
demonstration that the actions of oxidative agents on membrane currents
and conductance properties of RPE cells can be prevented by
antioxidants. Exposure of human RPE cells to glutathione precursors or
overexpression of the antioxidant enzyme GST in these cells diminished
the inhibitory effects of H2O2 on membrane
chloride conductances. These results suggest that the membrane chloride conductance in human RPE cells is modulated by reactive oxygen species
and can be protected by antioxidative agents.
Chloride channel expression.
In the present study, RT-PCR analysis of primary cultures of haRPE
cells resulted in the amplification of cDNA fragments with the
predicted size and sequence for several known ClC channels, specifically ClC-2, ClC-3, and ClC-5. These findings are similar to our
previous reports of ClC-5 and ClC-3 mRNA expression in cultured hfRPE
cells (39). Similar evidence for CFTR mRNA expression was
obtained in the same cells. These results agree with previous preliminary reports of CFTR mRNA expression in human RPE cells by
others (4, 30).
The results of Western blot analysis and immunocytochemical experiments
revealed positive reactivity with ClC channel antibodies that was
blocked by antigenic peptides. These findings indicate the expression
of ClC channel proteins in addition to mRNA expression. We previously
reported (39) ClC-2, ClC-3, and ClC-5 protein expression
in subconfluent fetal RPE cells. However, unlike the diffuse
intracellular staining noted in fetal cultured cells, the staining of
ClC-3 and ClC-5 in intact haRPE was predominantly located near the
region of the apical membrane.
Recent studies in other organs such as the kidney and liver indicate
that ClC-5 channels are mainly expressed in intracellular compartments
(for review see Ref. 38). Although the channels were found
in the apical region of the RPE cells in the present study, we cannot
rule out an intracellular location. It is conceivable that channels
could be contained both in the apical membrane and in a subcellular
compartment below the apical surface. Schwake et al. (31)
recently reported that ClC-5 can be inserted into surface membranes and
is retrieved from the surface membranes via a mechanism that involves
protein-protein interactions. These interactions are mediated by a PY
motif that, when mutated, abolishes retrieval of this channel from cell
surface membranes. Further work is needed to determine whether these
channels are trafficked to the apical membranes of RPE cells or are
involved in phagocytosis or endocytosis.
ClC-2 and ClC-3 are widely expressed in most epithelial cells
(38). Although strong staining was obtained for ClC-3
antibody, in preliminary experiments, ClC-2 immunoreactivity in haRPE
was weak. Nonetheless, ClC-2 expression was recently confirmed in mouse
RPE (6). Although ClC-3 is generally believed to be an intracellular ion channel (34), positive staining for
ClC-3 was observed in the apical membrane region of haRPE cells and this staining was blocked by antigenic peptide.
Recently, controversy has arisen concerning the specificity of ClC-3
antibodies. Stobrawa et al. (34) reported that ClC-3 antibody from Alomone Laboratories demonstrated positive reactivity against an unidentified peptide in ClC-3 knockout mice. We cannot rule
out the possibility that an unrelated peptide may have reacted with the
ClC-3 antibody used in the present study. However, in Western blot
studies, a positive reaction was obtained at the appropriate size for
ClC-3. In addition, as noted above, PCR analysis provides evidence for
mRNA expression for ClC-3. We note also that ClC-3 has been cloned from
human fetal cells (39) and intact human adult retina
(5).
As discussed above, CFTR expression in haRPE has been previously
reported in preliminary studies by Quong and Miller (30). Peterson et al. (26) reported bright staining of CFTR
predominantly near the basolateral membrane. In the present study,
staining near both membranes was detected. Further experiments are
needed to resolve this discrepancy and localize this channel.
Nonetheless, the results of the present study are consistent with CFTR
protein expression in haRPE cells and extend our previous findings of CFTR protein expression in cultured fetal RPE cells (39).
Chloride channel function.
Previous functional studies of human RPE have identified two
functionally distinct chloride channels located in the basolateral membrane, a cAMP-regulated channel and a calcium-regulated channel (28). In the present study, whole cell patch-clamp
measurements in primary cultures of haRPE cells demonstrated an
outwardly rectifying current that was reduced by replacement of
chloride in the bathing solution with iodide or gluconate. In addition,
the current was decreased by the chloride channel blockers chlorotoxin
and DIDS. The effects of calcium stimulation were not evaluated;
however, the current was increased by cAMP. These properties are nearly identical to those previously reported for cultured SV40-transformed hfRPE cells (39).
At present, the relationship between regulated membrane chloride
conductances in subconfluent RPE cells and specific ClC channels or
CFTR channels expressed in RPE cells is unclear. Unfortunately, it was
not possible to study the conductance properties of intact sheets of
haRPE in the present study. In the case of CFTR, it is generally
believed that this channel is exclusively expressed on the apical
membrane in CF-affected airway epithelia (26). However,
previous reports of a cAMP-stimulated conductance (28) and
the presence of CFTR near or in the basolateral membrane in RPE cells
(26) raise the possibility that CFTR may at least partly
mediate the basolateral membrane chloride conductance.
The putative apical and/or intracellular location of ClC-5 makes the
participation of these channels in the basolateral membrane conductance
unlikely. In other epithelial tissues such as the renal proximal tubule
and hepatocytes, these channels are located in endosomes beneath the
apical surface (8, 21, 31). In mice genetically engineered
to be deficient in this channel, apical membrane endocytosis is
impaired in proximal tubule cells but not for the sinusoidal membranes
of hepatic cells (27). However, the function of these
channels in endosomes is not well understood. Because the apical
membrane of RPE undergoes a substantial amount of remodeling and
endocytosis during phagocytosis, it would be interesting to determine
whether the RPE shows defective endocytosis in ClC-5-deficient mice.
ClC-3-deficient mice show normal retinas at birth, but a striking
photoreceptor degeneration occurs within the first 2 wk of life
(34). The reasons for this loss are not understood. Previous investigators have suggested that ClC-3 may be a
swelling-activated channel (9); however, no defects in
swelling activated conductances were found in ClC-3-deficient mice
(34). Moreover, Li et al. (20) demonstrated
that the properties of ClC-3 currents expressed in heterologous systems
were distinct from swelling activated currents. Therefore, ClC-3
channels may play a different, but presently unknown, role in the
maintenance of photoreceptor viability.
Oxidative stress and antioxidants.
Oxidative stress may play a role in RPE aging and in retinal
degenerative diseases (2); however, the mechanisms by
which oxidants cause damage to RPE and other epithelial cell membranes remain unclear (25). Addition of 100 µM
H2O2 resulted in reversible inhibition of the
membrane chloride conductance in primary cultures of haRPE cells. We
recently demonstrated (37) that similar concentrations of
H2O2 inhibit ClC-5 currents expressed in
mammalian somatic cells or Xenopus oocytes and membrane
chloride conductances in SV40-transformed hfRPE cells. CFTR is also
known to be affected by oxidative agents (18, 35). These
findings raise the possibility that membrane chloride channels,
possibly including CFTR and ClC-5, may be modulated in RPE cells by
redox-sensitive mechanism(s).
Role of antioxidants.
Antioxidants are known to protect cells from the deleterious effects of
oxidative stress. In previous studies, exposure of RPE cells to
H2O2 was found to induce mitochondrial DNA
damage (1) and apoptosis (15) and to
produce an increase in the activity of the antioxidant enzyme GST
(32). Preliminary studies using hfRPE cells transfected to
overexpress GST A1.1 showed protection from oxidant-induced DNA damage
and loss of cell viability (B. F. Godley, unpublished
observations). Using the same stably transfected cell line, we observed
that GST overexpression protects against the immediate inhibitory
effects of H2O2 on the membrane chloride conductances. Moreover, untransfected SV40-transformed hfRPE cells in
the present study were also protected against the inhibitory effects of
H2O2 on membrane chloride currents and
conductances when they were grown in media supplemented with precursors
of glutathione synthesis according to the protocol of Sternberg et al.
(33). It will be interesting to determine whether other oxidizing agents have similar inhibitory effects on membrane properties and, if so, whether other antioxidant mechanisms can protect against this inhibition.
In summary, haRPE cells express ClC and CFTR chloride channels,
suggesting that the role of these channels is not limited to fetal
development. In addition, these cells demonstrate a membrane chloride
conductance that is sensitive to cAMP and inhibited by the oxidant
H2O2. Overproduction of the antioxidant enzyme
GST or exposure to amino acids that are precursors to glutathione production protected against the effects of
H2O2. The deleterious effects of oxidants on
the membrane chloride conductance and the protective effects of
antioxidant systems raise the possibility that chloride channel
function in RPE cells may be altered in diseases or conditions that
increase oxidative damage such as diabetic retinopathy
(19) or macular degeneration (2). Such disruptions in RPE chloride channel functions could provide one means
by which oxidative stress contributes to pathophysiologies associated
with the retinal-RPE interface.
 |
ACKNOWLEDGEMENTS |
We are indebted to Drs. O. Devuyst and T. Jentsch for their
donation of ClC-5 and ClC-3 antibodies, to Dr. Alan Marmorstein for
donation of haRPE protein and help with the Western blot analyses, and
to S. Blaug for helpful suggestions regarding CFTR primers.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants
DK-53352, EY-12850, EY-11308, and EY-1792, a RPB Sybil B. Harrington
Scholar Award (B. F. Godley), and the John Sealy Memorial Endowment for Biomedical Sciences (N. K. Wills).
Address for reprint requests and other correspondence:
N. K. Wills, Depts. of Physiology and Biophysics and
Ophthalmology and Visual Sciences, Univ. of Texas Medical Branch,
Galveston, TX 77555
0641 (E-mail:
nkwills{at}utmb.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.
June 13, 2002;10.1152/ajpcell.00445.2001
Received 17 September 2001; accepted in final form 1 May 2002.
 |
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