Immunolocalization of electrogenic sodium-bicarbonate
cotransporters pNBC1 and kNBC1 in the rat eye
Dean
Bok1,2,
Matthew J.
Schibler2,
Alexander
Pushkin3,
Pejvak
Sassani3,
Natalia
Abuladze3,
Zarah
Naser4, and
Ira
Kurtz3
1 Jules Stein Eye Institute, Department of Neurobiology,
2 Brain Research Institute, 3 Division of Nephrology,
Center for Health Sciences, University of California Los Angeles School
of Medicine, Los Angeles, 90095-1689; and 4 Innovex Biosciences,
Richmond, California 94805
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ABSTRACT |
The human NBC1 gene encodes two
electrogenic sodium-bicarbonate cotransport proteins, pNBC1 and kNBC1,
which are candidate proteins for mediating electrogenic
sodium-bicarbonate cotransport in ocular cells. Mutations in the coding
region of the human NBC1 gene in exons common to both pNBC1 and kNBC1
result in a syndrome with a severe ocular and renal phenotype
(blindness, band keratopathy, glaucoma, cataracts, and proximal renal
tubular acidosis). In the present study, we determined the pattern of
electrogenic sodium-bicarbonate cotransporter protein expression in rat
eye. For this purpose, pNBC1- and kNBC1-specific antibodies were
generated and used to detect these NBC1 protein variants by
immunoblotting and immunocytochemistry. pNBC1 is expressed in cornea,
conjunctiva, lens, ciliary body, and retina, whereas the expression of
kNBC1 is restricted to the conjunctiva. These results provide the first
evidence for extrarenal kNBC1 protein expression. The data in this
study will serve as a basis for understanding the molecular mechanisms
responsible for abnormalities in ocular electrogenic sodium-bicarbonate
cotransport in patients with mutations in the NBC1 gene.
bicarbonate; sodium; transport; eyes; cornea; ciliary body; retina
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INTRODUCTION |
THE SODIUM-BICARBONATE
COTRANSPORTERS (NBCs) are a family of integral membrane proteins
that mediate electrogenic or electroneutral sodium-bicarbonate
cotransport (1, 2, 4, 12, 29, 46, 47, 51). Members of the
NBC protein family play an important role in intracellular pH
regulation and transepithelial transport of sodium and bicarbonate in
several tissues. In various cell types in the eye, electrogenic
sodium-bicarbonate cotransport plays an important role in intracellular
pH regulation and transepithelial ion flux. The importance of
electrogenic sodium-bicarbonate cotransport to normal ocular function
is highlighted by recent evidence that patients with homozygous
mutations in the NBC1 (SLC4A4) gene have an ocular phenotype
characterized by blindness, glaucoma, cataracts, and band keratopathy
(28). Extraocular manifestations include severe
proximal renal tubular acidosis, mental retardation, short stature, an
elevated serum amylase, and thyroid abnormalities. The cellular
mechanism(s) responsible for the ocular abnormalities in these patients
is presently not known. Characterization of the expression pattern of
NBC1 proteins in the eye would improve our understanding of the
molecular pathogenesis of the cause of blindness in this disorder.
There are several cell types in the eye where functional electrogenic
sodium-bicarbonate cotransport has been reported. Electrogenic sodium-bicarbonate cotransport has been described in corneal
endothelial cells (10, 30), pigmented ciliary body
epithelium (64), lens epithelium (62),
retinal astrocytes and Müller cells (42-45), and retinal pigment epithelium (26, 31, 35). These studies have utilized several different model systems including intact epithelial preparations, cultured cells, and vesicle preparations from
various species. Depending on the specific cell type, electrogenic bicarbonate transport is thought to play an important role in mediating
transepithelial sodium, bicarbonate, and fluid transport (e.g., corneal
endothelial cells, ciliary body epithelium, retinal pigmented
epithelium), intracellular pH regulation, and maintenance of
extracellular pH (retinal astrocytes and Müller cells). Ion and
fluid transport in these tissues are thought to play an important role
in the control of corneal transparency, lens transparency, intraocular
pressure, retinal function, and attachment.
In humans, electrogenic sodium-bicarbonate cotransport is
mediated by kNBC1 and pNBC1 proteins, which are encoded by the NBC1 gene (2). kNBC1 and pNBC1 are highly homologous proteins
that have different NH2 termini, in that in kNBC1 41 amino
acids replace the initial 85 amino acids of pNBC1 (1).
pNBC1 is highly expressed in the pancreas, where it is thought to play
an important role in mediating basolateral bicarbonate influx in ductal
cells and resultant transepithelial bicarbonate secretion, whereas
kNBC1 is thought to play an important role in mediating renal proximal tubular bicarbonate absorption (1, 12, 51, 52). The
expression pattern of these transporter proteins in various cells in
the eye has not been characterized. Knowledge of the cellular
distribution of electrogenic sodium-bicarbonate cotransport proteins in
ocular tissues would begin to provide a molecular basis for
understanding the potential role of loss of function mutations in pNBC1
and kNBC1 in causing blindness (28). In a recent study,
Sun et al. (56) reported the presence of NBC1 on the
basolateral membrane of corneal endothelial cells. However, there are
presently no studies that have utilized specific antibodies to
determine the tissue expression pattern of pNBC1 and kNBC1 proteins. To
gain further insight into the molecular mechanisms responsible for electrogenic sodium and bicarbonate transport in the eye, we generated rabbit polyclonal antibodies against the unique NH2
terminus of pNBC1 and kNBC1 and determined the cellular localization of
each protein in the rat eye. A comprehensive analysis of the expression of pNBC1 and kNBC1 in various ocular tissues is reported.
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METHODS |
Generation and characterization of polyclonal antibodies to pNBC1
and kNBC1.
A polyclonal antibody to pNBC1 was raised in rabbits against a
synthetic peptide derived from the NH2 terminus of the
protein (amino acids 1-19, coupled to an NH2-terminal
cysteine). The affinity-purified polyclonal antibody to kNBC1 was
raised against a synthetic peptide, corresponding to amino acids
11-24, coupled to an NH2-terminal cysteine. Both
antibodies were affinity purified using Sepharose 4B columns with
covalently attached pNBC1 or kNBC1 peptides. To characterize the
antibodies, HEK-293T cells grown on fibronectin-coated coverslips were
transfected using the calcium phosphate precipitation method with
plasmids (pcDNA3.1, Invitrogen, Carlsbad, CA) containing the coding
region for pNBC1 or kNBC1. The cells were rinsed in methanol at
20°C for 5 min and washed in PBS several times. Each primary
antibody (1:100 dilution) was applied for 1 h and, after several
washes in PBS, goat anti-rabbit IgG conjugated with Alexa 594 (1:500
dilution, Molecular Probes, Eugene, OR) was applied for an additional
1 h. The slides were washed in PBS and mounted in Cytoseal 60 (Stephens Scientific, Riverdale, NJ). A liquid-cooled PXL
charge-coupled device camera (model CH1, Photometrics, Osnabruck, Germany), coupled to a Nikon Microphot-FXA epifluorescence microscope, was used to capture and digitize the fluorescence images of the cells
expressing pNBC1 or kNBC1. The images were transferred to a Silicon
Graphics Indy 5000 computer using ISEE 4.0 (c) software (Inovision,
Raleigh, NC).
Western blotting.
The eyes from normal Long-Evans rats were divided into cornea, ciliary
body, conjunctiva, and retina. These tissues were disrupted at 0°C in
a glass homogenizer with 20 mM Tris · HCl, pH 7.5, containing 140 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 µg/ml
pepstatin, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. The homogenate
was centrifuged at 300 g for 10 min, and the supernatant was
analyzed by SDS-PAGE. Membranes from rat pancreas and kidney were
isolated using differential centrifugation. The proteins were separated
by SDS-PAGE and electrotransferred onto a polyvinylidene difluoride
membrane (Bio-Rad, Hercules, CA). Nonspecific binding was
blocked by incubation of the membrane in 20 mM Tris · HCl, pH
7.5, 140 mM NaCl, and 0.1% Tween 20 (TBST) containing 5% dry milk.
Primary pNBC1- or kNBC1-specific antibodies were used at a dilution of
1:1,000. Secondary horseradish peroxidase-conjugated mouse anti-rabbit
antibody (Jackson ImmunoResearch, West Grove, PA) was used at a
dilution of 1:10,000. The bands were visualized using an enhanced
chemiluminescence (ECL) kit and ECL Hyperfilm (Amersham Pharmacia
Biotech, Keene, PA).
Immunocytochemistry.
For immunofluorescence experiments, rat eyes were removed and
immediately frozen in dry ice. The pNBC1 and kNBC1 primary antibodies (1:100 dilution) were applied to 5-µm cryostat sections for 45 min
and, after several washes in PBS, goat anti-rabbit IgG conjugated with
Alexa 594 (1:500 dilution, Molecular Probes) was applied for 40 min. In
control experiments, the primary antibodies were preabsorbed with the
specific immunizing peptide (10 µg/ml). The slides were washed in PBS
and mounted in Cytoseal 60 (Stephens Scientific). The confocal images
were captured with a Leica TCS SP inverted confocal microscope (Leica)
using a krypton laser (model 643, Melles Griot, Irvine, CA). In some
experiments, rat eyes were dissected and immersed immediately in PBS
with 4% paraformaldehyde. After fixation for 24 h at 4°C, the
samples were washed in PBS, dehydrated through a graded series of
ethanol washes, cleared in xylene, and embedded in paraffin. Sections
were cut at a thickness of 7 µm, mounted onto glass slides, and
stored at room temperature until use. The slides were deparaffinized in
graded degrees of ethanol and treated with xylene. The sections were
immersed in freshly made 3% hydrogen peroxide prepared in water for 30 min and then rinsed in water. The pNBC1 and kNBC1 primary antibodies (1:100 dilution) were applied for 20 min and, after a 5-s wash, were
incubated in a biotinylated anti-rabbit secondary antibody (STAT-Q,
Innovex Biosciences, Richmond, CA) for 10 min. The sections were then
incubated in peroxidase-labeled strepavidin for 10 min, immersed in a
diaminobenzidine substrate solution for 5 min, and subsequently
counterstained with hematoxylin and mounted.
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RESULTS |
To establish the presence and examine the cellular distribution of
pNBC1 and kNBC1 in the rat eye, rabbit affinity-purified polyclonal
antibodies were raised against the specific NH2 terminus of
each protein. The polyclonal antibodies against pNBC1 and kNBC1 were
initially characterized by transfection experiments in HEK-293T cells.
As shown in Fig. 1, immunofluorescence
microscopy revealed that pNBC1 and kNBC1 are readily detected in cells
expressing each protein. No labeling was observed in untransfected
cells. Labeling of the pancreas with the anti-pNBC1 antibody and of the kidney with the anti-kNBC1 antibody are also shown.

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Fig. 1.
A-D: immunocytochemical analysis of the
sodium-bicarbonate cotransporter expression of pNBC1 and kNBC1 in
HEK-293T cells. A: transfected cells labeled with the
anti-pNBC1 antibody, showing a predominantly plasma membrane
localization of pNBC1. These cells did not label with the anti-kNBC1
antibody. B: untransfected cells labeled with the anti-pNBC1
antibody. C: transfected cells labeled with the anti-kNBC1
antibody, showing a predominantly plasma membrane localization of
kNBC1. These cells did not label with the anti-pNBC1 antibody.
D: untransfected cells labeled with the anti-kNBC1 antibody.
E: overlapping Nomarski and immunofluorescence image showing
labeling of pancreatic ducts with the anti-pNBC1 antibody.
F: immunofluorescence staining of proximal tubules in the
kidney with the anti-kNBC1 antibody.
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The pNBC1 antibody recognized a ~145-kDa band on immunoblots of rat
cornea, ciliary body, retina, and conjunctiva (Fig.
2). The specificity of the labeling was
confirmed using the pNBC1 antibody preabsorbed with the immunizing
peptide. In addition, data from rat pancreatic membranes that strongly
express pNBC1 are shown for comparison. The size of the recognized
protein in all tissues is larger than the predicted size of ~121 kDa,
suggesting that pNBC1 is glycosylated or posttranslationally modified
in other ways.

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Fig. 2.
Immunoblots from various rat ocular tissues. A:
lanes 1 and 6: cornea; lanes 2 and 7: ciliary body; lanes 3 and 8: retina;
lanes 4 and 9: conjunctiva; and lanes 5 and
10: pancreas. Loading: lanes 1-4 and
6-9: 80 µg; lanes 5 and 10: 20 µg. Lanes 1-5 were probed with the anti-pNBC1
antibody; lanes 6-10 were probed with the anti-pNBC1
antibody preincubated with 10 µg/ml of specific immunizing peptide.
B: lanes 1 and 6: cornea; lanes
2 and 7: ciliary body; lanes 3 and
8: retina; lanes 4 and 9: conjunctiva;
lanes 5 and 10: kidney. Loading: lanes 1-4
and 6-9: 80 µg; lanes 5 and
10: 40 µg. Lanes 1-5 were probed with the
anti-kNBC1 antibody; lanes 6-10 were probed with the
anti-kNBC1 antibody preincubated with 10 µg/ml of specific immunizing
peptide.
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Unlike pNBC1, kNBC1 was found in conjunctiva only. As shown in Fig. 2,
the kNBC1 antibody recognized a ~130-kDa band in conjunctiva only.
Furthermore, kNBC1 was not detectable in cornea, ciliary body, or
retina. These findings suggest that kNBC1 plays a more specialized role
in mediating electrogenic sodium-bicarbonate cotransport in the
conjunctiva. Given a predicted size of ~116 kDa, the results indicate
that the kNBC1 is posttranslationally modified. In an immunoblot of rat
kidney membranes known to strongly express kNBC1, a band of ~130 kDa
was also detected and is illustrated in Fig. 2 for comparison.
pNBC1 immunostaining was observed in many ocular tissues, and in a
cell-specific pattern. Figure 3 depicts
the immunolocalization of pNBC1 in the cornea. In the cornea,
epithelial cells in the basal and intermediate cell layers were stained
most intensely, with only occasional cells staining in the surface
layer (Fig. 3, A-D). Cells in the basal and
intermediate layers in the peripheral cornea (Fig. 3A) were
more uniformly stained than those of the central cornea (Fig. 3,
B and D). Within the central cornea, some of the
cells in the basal layer were not labeled (Fig. 3, B and D). There was no clearly defined preferential staining of
apical and basolateral membrane domains in the basal layer or
elsewhere. As shown in Fig. 3, A, B, and
D, keratocytes of the corneal stroma were also stained for
pNBC1. Unlike pNBC1, kNBC1 was not detected in corneal epithelial cells
(Fig. 3, E and F). In the corneal endothelium
(Fig. 4), pNBC1 was predominantly
localized to the basolateral cell membrane of endothelial cells, as
shown in Fig. 4C. Corneal endothelial cells also failed to
stain with the anti-kNBC1 antibody (Fig. 4, E and
F).

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Fig. 3.
A-F: immunolocalization of pNBC1 in rat corneal
epithelium and stroma. A and B: immunoperoxidase
labeling of paraffin-embedded rat cornea. A: peripheral
cornea showing the plasma membrane of epithelial cells in the basal
layer labeled with the anti-pNBC1 antibody (arrows). Bar, 46 µm.
B: the central cornea showing cells in the basal layer
staining for pNBC1. Note that epithelial cells in this region of the
cornea were less frequently labeled (arrows). Bar, 45 µm. In addition
to epithelial cells, stromal keratocytes were also labeled (arrowheads,
A and B). C: Nomarski image of cornea
showing the corneal epithelium and stroma. Bar, 35 µm. D:
immunofluorescence staining of cryostat cornea sections showing
epithelial cells in the basal, intermediate, and surface layer labeled
with the anti-pNBC1 antibody (arrows). Labeling of stromal keratocytes
with the anti-pNBC1 antibody is also shown (arrowheads). Bar, 35 µm.
E: Nomarski image of cornea depicting the epithelial layer
and stroma. Bar, 35 µm. F: specific peptide blocking of
pNBC1 staining. Bar, 35 µm.
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Fig. 4.
Immunolocalization of pNBC1 (A-D) and kNBC1
(E and F) in corneal endothelium. A:
Nomarski image of the cornea depicting the stroma and corneal
endothelial layer. Bar, 35 micros B: immunofluorescence
staining of the corneal endothelial cells labeled with the anti-pNBC1
antibody (arrow). Bar, 35 µm. C: high-power image of
corneal endothelial cells. Overlapping Nomarski and immunofluorescence
image showing labeling of endothelial cells using the anti-pNBC1
antibody (arrows). Bar, 9 µm. D: specific peptide blocking
of pNBC1 staining. Bar, 35 µm. E: Nomarski image of cornea
showing the corneal epithelium, stroma, and endothelium. Bar, 35 µm.
F: immunofluorescence image showing the lack of any cell
staining in the cornea with the anti-kNBC1 antibody. Bars, 35 µm.
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In contrast to the cornea, pNBC1 was not detected in the iris (Fig.
5, A and B). The
posterior pigmented epithelium, the dilator iridis muscle, the stroma,
and the anterior border layer were not stained with anti-pNBC1
antibody. Similarly, kNBC1 was undetectable in the iris (Fig. 5,
C and D). In addition, components of the outflow
pathway for aqueous humor such as the trabecular meshwork and the canal
of Schlemn were not stained with either antibody.

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Fig. 5.
Nomarski (A and C) and corresponding
immunofluorescence images (B and D) showing lack
of staining of the iris with either the anti-pNBC1 antibody
(B) or the anti-kNBC1 antibody (D). The canal of
Schlemn (arrow) depicted in the Nomarski image (E) was not
stained with the anti-pNBC1 antibody (F). Bars, 35 µm
(A-D); 26 µm (E and
F).
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As shown in Fig. 6, the lens epithelium
was stained uniformly from the anterior pole and extended posteriorly
to the region of lens fiber cell differentiation. Apical and
basolateral plasma membranes were labeled with the anti-pNBC1 antibody.
kNBC1 was not detectable (Fig. 6, E and F).

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Fig. 6.
The lens epithelium (arrows) was labeled with the
anti-pNBC1antibody. Shown here is the anterior epithelium labeled with
the anti-pNBC1 antibody, however, the entire epithelium extending to
the equatorial zone of differentiation into lens fiber cells was also
labeled. A: Nomarski image of the lens showing the capsule,
lens fiber cells, and epithelium. Bar, 35 µm. B:
immunofluorescence image of the lens stained with anti-pNBC1. The
labeling was not polarized to a specific membrane domain. Bar, 35 µm.
C: overlapping Nomarski and immunofluorescence image. Bar,
35 µm. D: specific peptide blocking of pNBC1 staining.
Bar, 35 µm. E: Nomarski image. Bar, 35 µm. F:
immunofluorescence image showing the lack of staining in the lens with
the anti-kNBC1 antibody. Bar, 35 µm.
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The conjunctival epithelium was labeled by the anti-pNBC1 antibody
(Fig. 7) and by the anti-kNBC1 antibody
(Fig. 8); however, the staining patterns
were different. pNBC1 labeled surface and wing cells; however, kNBC1
was detected predominantly in basal cells. This is the first evidence
of kNBC1 protein expression in an extrarenal location. No labeling was
observed in the conjunctival stroma.

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Fig. 7.
Immunolocalization of pNBC1 (A-F) in the
conjunctiva. Nomarski image (A) and immunofluorescence image
(B) showing surface cells (arrow) and wing cells (arrowhead)
stained with the anti-pNBC1 antibody (A and B;
bar, 35 µm). C and D: high-power images of the
conjunctiva showing surface cells (arrow) and wing cells (arrowhead)
stained with the anti-pNBC1 antibody. Nomarski image (C) and
immunofluorescence image (D). C and D;
bar, 9 µm. Overlapping low-power (E; bar, 35 µm) and
high-power (F; bar, 9 µm) images showing the anti-pNBC1
antibody labeling of conjunctival epithelial cells.
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Fig. 8.
Immunolocalization of kNBC1 (A-F) in the
conjunctiva. A: immunofluorescence image showing basal
epithelial cells (arrow) stained with the anti-kNBC1 antibody. Bar, 35 µm. B: specific peptide blocking of kNBC1 staining. Bar,
35 µm. C: immunofluorescence image showing basal
epithelial cell kNBC1 staining (arrow), and overlapping
Nomarski/immunofluorescence image (D) of the conjunctiva.
Bar, 35 µm. High-power Nomarski image (E) and
immunofluorescence image (F) of the conjunctiva showing
kNBC1 basal epithelial cell membrane staining. (E and
F; Bars, 5.5 µm).
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The pigmented epithelium of the ciliary body processes was stained
intensely for pNBC1 (Fig. 9). Labeling
was present in the basolateral membrane of ciliary pigmented epithelial
cells. In contrast to the strong labeling of pigmented
epithelial cells, the anti-pNBC1 antibody did not label the
nonpigmented epithelial cell layer. Furthermore, the capillaries, the
postcapillary venules, and the ciliary stroma were unlabeled. As shown
in Fig. 9, E and F, kNBC1 was not detected in
either the pigmented or nonpigmented ciliary epithelium.

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Fig. 9.
Immunolocalization of pNBC1 (A-F) in the ciliary
body processes. A: Nomarski image showing a longitudinal
section of a ciliary body process. Bar, 35 µm. B:
immunofluorescence image showing staining of the basolateral membrane
of pigmented epithelial cells. Bar, 35 µm. C: overlapping
image showing unlabeled nonpigmented epithelial cells (arrows) and
basolateral membrane labeling of pigmented epithelial cells
(arrowheads). Bar, 35 µm. D: specific peptide blocking of
pNBC1 staining. Bar, 35 µm. E: Nomarski image of a
longitudinal section of a ciliary body process and the corresponding
immunofluorescence image (F) showing lack of staining with
the anti-kNBC1 antibody. Bar, 35 µm.
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Within the retina, anti-pNBC1 labeling was detected in both the
retinal pigment epithelium (RPE) and the neurosensory retina. As shown
in Fig. 10, A and
B, the apical membrane of RPE cells was labeled with the
anti-pNBC1 antibody. Furthermore, within the choroid and sclera,
occasionally labeled fibroblasts were detected (Fig. 10, C
and D). As shown in Fig. 10, E and F,
kNBC1 was not detected in either the RPE or the neurosensory retina. Within the neurosensory retina, Müller glial cells were labeled with the anti-pNBC1 antibody. As shown in Fig.
11, A-C, the
apical microvilli of Müller cells labeled strongly. Figure
11E shows Müller cell membrane staining in the inner
nuclear layer where the Müller cells pass among neurons. In the
nerve fiber layer, pNBC1 staining was detected in Müller cell end
feet (Fig. 11F). Therefore, pNBC1 staining of Müller
cells extended from the apical microvilli in the outer limiting
membrane and continued radially throughout the retina. The anti-pNBC1
antibody failed to label rod and cone photoreceptor cells and other
neural elements.

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Fig. 10.
Immunolocalization of pNBC1 in the retina, choroid, and sclera.
A: immunofluorescence image of the retinal pigment
epithelium labeled with the anti-pNBC1 antibody. Bar, 9 µm.
B: overlapping Nomarski/immunofluorescence image of the
retinal pigment epithelium and choroid showing localization of pNBC1 to
the apical membrane (arrows). Some cells were unlabeled. Occasional
fibroblasts in the choroid are also labeled (arrowheads). Bar, 9 µm.
C: high-power Nomarski image and corresponding
immunofluorescence image (D) showing fibroblasts in the
choroid and sclera labeled with the anti-pNBC1 antibody. Bar
(C and D), 9 µm. E: Nomarski image
of the neurosensory retina and the corresponding immunofluorescence
image (F) showing lack of staining with the anti-kNBC1
antibody. Bar (E and F), 35 µm.
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Fig. 11.
Labeling of Müller cells with the anti-pNBC1 antibody.
A: Nomarski image and corresponding immunofluorescence
(B) and overlapping image (C) showing pNBC1
staining of the apical microvilli (arrows) and the processes
(arrowheads) of Müller cells surrounding the nuclei of
photoreceptors. Bar (A-C), 10 µm. D:
specific peptide blocking of pNBC1 staining in the region shown in
A-C. Bar, 10 µm. E: overlapping
Nomarski/immunofluorescence image of Müller cell bodies and their
processes in the inner nuclear layer. Bar, 10 µm. F:
immunofluorescence image of Müller cell end feet at the vitreal
surface of the retina. Bar, 35 µm.
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DISCUSSION |
In the present study, we report the
immunolocalization of pNBC1 and kNBC1 proteins in the rat
eye using antibodies specific for each protein (summarized in Fig.
12). The results demonstrate that pNBC1 is widely distributed in the eye in various cell types including cornea, conjunctiva, lens epithelium, ciliary body, and
retina. In contrast, kNBC1 was only expressed in the basal epithelial
cells of the conjunctiva. The widespread localization of pNBC1 was
documented in immunoblots of cornea, conjunctiva, ciliary body, and
retina, which confirmed the presence of a ~145-kDa band corresponding
to pNBC1 in these tissues. The results suggest that the electrogenic
sodium-bicarbonate cotransporter pNBC1 plays an important physiological
role in several cell types in the eye. In contrast, a ~130-kDa band
corresponding to kNBC1 was only detected in conjunctiva. kNBC1 is
localized in the basolateral membrane of the renal proximal tubule,
where it is known to play an important role in mediating basolateral
sodium-bicarbonate efflux. The finding that kNBC1 protein is also
expressed in the basal epithelial cells of the conjunctiva is the first
suggestion that this NBC1 variant plays a physiological role in
extrarenal cells.
In pancreatic ductal cells, pNBC1 is expressed on the basolateral
membrane, where it mediates electrogenic sodium-bicarbonate influx with
a 2 HCO
:1 Na+ stoichiometry
(18). In the proximal tubule, kNBC1 has a stoichiometry of
3 HCO
:1 Na+ and mediates cellular
bicarbonate efflux because the reversal potential of the transporter is
less negative than the resting membrane potential (69). We
have recently demonstrated that both pNBC1 and kNBC1 can function in
either a 2 HCO
:1 Na+ or 3 HCO
:1 Na+ mode depending on the cell
type in which each transporter is expressed, indicating that an unknown
cellular factor(s) can alter the stoichiometry of both transporters
(19). These findings suggest the intriguing possibility
that the various cell types in the eye expressing pNBC1 may exhibit
differences in HCO
:Na+ stoichiometry. In
keeping with this possibility, RPE and retinal Müller cells each
express pNBC1, and yet the HCO
:Na+
stoichiometry appears to be 2:1 in the RPE (26) and 3:1 in retinal Müller cells (43). Therefore kNBC1 may not
necessarily exhibit a 3 HCO
:1 Na+
stoichiometry in conjunctival basal epithelial cells. Whether a
difference in the function or regulation of pNBC1 and kNBC1 necessitates the unique expression pattern of each transporter in
conjunctival cells requires further study.
Previous studies have demonstrated that the corneal endothelium
transports bicarbonate ions from the stroma to the surrounding aqueous
humor (22, 25). Sodium and bicarbonate play an important role in maintaining corneal hydration (34, 49, 50) and in endothelial cell intracellular pH regulation (56, 59).
Although electrogenic sodium-bicarbonate cotransport has been
implicated in this process, these studies have been based on cultured
cells, which may express proteins that are not necessarily present in corneal endothelial cells in vivo. In contrast to experiments utilizing
cultured endothelial cells, studies of vesicles derived from native
corneal endothelial cells have failed to demonstrate electrogenic
sodium-bicarbonate cotransport (36-38). Indeed, it has been postulated that a novel chloride-dependent electrogenic sodium- and bicarbonate-dependent transporter may mediate corneal endothelial bicarbonate efflux (38). Furthermore, these
authors have recently reported their failure to detect pNBC1 and kNBC1 transcripts in freshly isolated corneal endothelial cells
(38). The latter results are in contrast to a recent
report showing pNBC1 and kNBC1 transcripts in cultured human corneal
endothelial cells (59), and a separate study, which
demonstrated that NBC1 protein is expressed in freshly dissected and
cultured bovine corneal endothelial cells (56). However,
the antibody used in the latter study was unable to distinguish which
NBC1 variant, pNBC1 or kNBC1, is present. With the use of antibodies
that are specific for each NBC1 variant, the results in the present
study definitively demonstrate that pNBC1 protein is localized to the basolateral membrane of corneal endothelial cells in vivo, where it
likely plays an important role in mediating endothelial cell basolateral sodium-bicarbonate cotransport. Furthermore, kNBC1 protein
is not expressed in any cells in the cornea.
Of interest, in addition to endothelial cells, corneal epithelial cells
also expressed pNBC1, with cells in the basal and intermediate layers
expressing this protein most frequently. Unlike corneal endothelial
cells, which expressed pNBC1 in a polarized manner, the entire plasma
membrane of corneal epithelial cells was stained. There is presently
limited information on the transport properties of corneal epithelial
cells, despite their importance in functioning as a barrier against the
environment and in forming, along with the tear film, a refractive
surface that is essential for visual acuity. Recent studies have
identified a nonselective cation current, an outwardly rectifying
K+ current, a voltage-gated Na+ current, and
inwardly rectifying K+ currents in cultured human corneal
epithelial cells (9). However, there is presently no
information on the acid-base transport properties of corneal epithelial
cells, nor information regarding transport mechanisms responsible for
pH regulation. Interestingly, the PCO2 in
corneal epithelial cells varies from ~5 Torr in the open eye to 55 Torr in the closed eye (16). This variation in the
carbonic acid load requires potent acid-base transporters to maintain
intracellular pH in the physiological range. The finding that pNBC1 is
expressed in corneal epithelial cells suggests that it may play an
important role in regulating intracellular pH in this cell type, which
potentially contributes to the maintenance of corneal transparency.
The lens possesses an epithelial cell layer that underlies the anterior
capsule. Ion transport by these cells has been suggested to play an
important role in controlling the hydration and transparency of the
lens (7, 8, 13, 41). Measurement of unidirectional fluxes
indicates that 35% of the translenticular short-circuit current is
mediated by active sodium transport (13). The toad lens
epithelium has been shown to have an electrogenic sodium-bicarbonate cotransporter that appears to function with a 2:1 stoichiometry (62). Although there are no studies that have
characterized this transporter in the mammalian lens, our results
suggest that pNBC1 mediates electrogenic sodium-bicarbonate cotransport
in this part of the eye. Interestingly, a recent study indicates that
-crystallin binds lens fiber membranes through hydrophobic interactions that are strikingly pH and ionic strength dependent (14). The amount of
-crystallin that binds to the
membrane increases under acidic pH conditions (14).
Patients with loss-of-function pNBC1 mutations may have cataracts
because of an alteration in the pH or ionic strength near lens fiber cells.
The conjunctival epithelium has been shown to have significant
ion-transporting capabilities and is likely important, along with
conjunctival goblet cells, in the formation and/or modification of the
tear film (24, 54, 55, 60). Sodium, chloride, and glucose
have been shown to be actively transported across the conjunctival
epithelium (23, 24, 54). The conjunctival epithelium mediates active chloride and water secretion subject to modulation by
cAMP, Ca2+, protein kinase C, and purine and pyrimidine
nucleotides (3, 23, 24, 33, 58). The present model of
electrolyte transport in conjunctival cells includes an apical membrane
sodium-glucose cotransporter and a cAMP-stimulated chloride channel
(23, 33). The Na+-K+-ATPase and a
bumetinide-sensitive Na+-K+-2Cl
cotransport process are localized to the basolateral membrane (32). Previous studies have demonstrated that aquaporin-3
is also expressed uniquely in the conjunctival epithelium
(21). The relative proportion of tear volume attributable
to fluid secreted by the conjunctiva is not known. The results of the
present study suggest that H+/base transport may play a
heretofore unrecognized role in tear film formation/modification. pNBC1
protein was localized to surface and wing cells in the conjunctiva. No
labeling was observed in the conjunctival stroma. The finding that
kNBC1 was localized uniquely to basal cells in the conjunctiva suggests
an important role for this protein in transporting sodium and
bicarbonate in this cell type. It has been postulated that conjunctival
fluid secretion plays an important role in hydrating mucus secreted by
goblet cells (55). Impaired NBC1-mediated sodium transport could secondarily affect passive chloride uptake via the basolateral Na+-K+-2Cl
cotransporter and
subsequent apical chloride secretion, thereby altering transepithelial
fluid secretion. Whether conjunctival fluid secretion is abnormal in
patients with mutations in the NBC1 (SLC4A4) gene requires further investigation.
pNBC1 was immunolocalized to the basolateral membrane of pigmented
ciliary epithelial cells. The ciliary body is comprised of two cell
layers (pigmented and nonpigmented), whose apical cell membranes are
juxtaposed. Secretion of aqueous humor is thought be mediated by the
transfer of ions across the basolateral membrane of the pigmented
epithelial cell layer, crossing through gap junctions linking the
pigmented and nonpigmented cells (42). Water and ions then
enter the aqueous humor across the basolateral membrane of the
nonpigmented epithelium, although paracellular transport has also been suggested (48). Several transport mechanisms
have been implicated in mediating the secretion of aqueous humor,
including Na+-K+-2Cl
cotransport,
Na+/H+ exchange,
Cl
/HCO
exchange, vacuolar
H+-ATPase, and sodium-bicarbonate cotransport (42,
61, 63, 64, 66, 67). However, it is not completely understood
how these transport mechanisms are integrated into a cell model of pigmented and nonpigmented epithelial cell transport to account for the
formation of aqueous humor. Furthermore, the complexity of the
dual-layered ciliary body epithelium imposes serious difficulties in
determining the molecular mechanisms responsible for regulation of
aqueous humor formation. The finding that pNBC1 protein is localized to
the basolateral membrane of pigmented epithelial cells suggests that
electrogenic sodium-bicarbonate cotransport plays an essential role in
mediating transepithelial transport/intracellular pH (pHi)
regulation in these cells and in the formation of aqueous humor. This
result is in keeping with the studies of Wolosin et al.
(64), who functionally identified a sodium- and
bicarbonate-dependent, apparently electrogenic, transport process in
the freshly dissected rabbit ciliary body-pigmented epithelium. In
contrast, in nonpigmented ciliary epithelial cells, bicarbonate
transport was mediated by a chloride-bicarbonate exchange process
(64). An understanding of the transport processes involved
in aqueous humor production is clearly clinically important, given that
inhibition of aqueous formation with subsequent lowering of intraocular
pressure is a common approach for treating open-angle glaucoma
(39, 65). Although carbonic anhydrase inhibitors are among
the most potent inhibitors of aqueous humor formation, their cellular
site of action is unclear. It has been established that carbonic
anhydrase inhibitors reduce not only the rate of aqueous humor
formation but also the net rate of sodium and bicarbonate transport
from blood to aqueous humor (40). Carbonic anhydrase
inhibitors can alter bicarbonate absorption in transporting epithelia
by decreasing the rate of hydration of CO2 and subsequent
generation of bicarbonate (39, 40, 65). Our results
suggest that inhibition of pNBC1-mediated bicarbonate uptake by
pigmented ciliary body epithelial cells is a potential mechanism for
inhibition of aqueous humor production by these drugs, which requires
further investigation.
Previous studies have documented that retinal activation by light
results in an increase in extracellular pH in the inner plexiform
(synaptic) layer and in the photoreceptor layer (11, 68).
This increase in extracellular pH is mediated by increased synaptic
activity, in addition to a reduction in the metabolic activity of
the photoreceptors. A change in intracellular or extracellular pH
can result in marked changes in neuronal function, including gap
junction conductance, N-methyl-D-aspartate
receptor behavior, and voltage-gated ion channel transport (5, 6,
17, 57). For example, previous studies have shown that an
increase in extracellular pH of only 0.05 pH units can increase
synaptic transmission between photoreceptors and bipolar cells by
~25% (6). Newman (45) has hypothesized
that bicarbonate uptake mediated by an electrogenic sodium-bicarbonate
cotransporter in retinal Müller cells may prevent the elevation
of extracellular pH in the inner plexiform (synaptic) layer and in the
photoreceptor layer. Our finding that pNBC1 protein is localized to
retinal Müller cells is compatible with Newman's hypothesis and
suggests that pNBC1-mediated Müller cell bicarbonate uptake plays
an important role in preventing the extracellular pH from increasing
during retinal light stimulation.
The RPE mediates the transport of fluid, ions, and metabolites between
the subretinal space and the choroidal blood supply and plays an
essential role in maintaining the viability of the neural retina
(27). In native RPE preparations, the cell transport model
includes an apical Na+-K+-ATPase,
Na+/H+exchange, electrogenic sodium-bicarbonate
cotransport, H+-lactate cotransport, and basolateral
Cl
/HCO
exchange (26, 27,
35). There is also recent evidence for a possible basolateral
membrane sodium-bicarbonate cotransport process (31).
These cells are normally exposed to lactic acid produced by
photoreceptors under aerobic conditions (20). pNBC1 likely
plays an important role in maintaining RPE intracellular pH constant,
despite H+ influx mediated by the apical
H+-lactate cotransporter. pNBC1 may also play a role in
mediating apical-to- basolateral bicarbonate transport in the bullfrog
RPE (26), although the relative importance of this
transport process in mammals appears to be species dependent
(15). In addition, pNBC1-mediated alterations in
intracellular pH in response to apical potassium concentration changes
may be an important signaling event after dark-light transition
(31).
The potential cause of blindness in patients with mutations in the NBC1
gene reported by Igarashi et al. (28) is clearly very
complex, given the various regions in the eye where NBC1 proteins are
localized. The patients reported by Igarashi et al. had mutations in
the coding region common to pNBC1 and kNBC1. Whether abnormal pNBC1
and/or kNBC1 function is responsible for the ocular phenotype in these
patients requires further study. However, it is likely, given our
findings of the widespread distribution of pNBC1 in the eye (cornea,
conjunctiva, lens, ciliary body, and retina) and the fact that kNBC1 is
only expressed in conjunctiva, that the phenotype reported in these
patients is most likely due to abnormal pNBC1-mediated
sodium-bicarbonate cotransport. Thus far, patients with mutations in
the unique NH2 terminus of pNBC1 or kNBC1 have not been
reported. It would be predicted that those patients with mutations in
the unique kNBC1 NH2 terminus would present with
predominantly a renal phenotype (proximal renal tubular acidosis), with
minimal ocular abnormalities involving conjuntival tear film
formation/modification. Knockout mice with specific NBC1 mutations will
help address these issues.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-46976, DK-58563, and DK-07789,
and the Iris and B. Gerald Cantor Foundation, the Max Factor Family
Foundation, the Verna Harrah Foundation, the Richard and Hinda
Rosenthal Foundation, and the Fredericka Taubitz Foundation. N. Abuladze is supported by training Grant J891002 from the National Kidney Foundation of Southern California.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: I. Kurtz, UCLA School of Medicine, Division of Nephrology, 10833 Le Conte
Ave., Rm. 7-155 Factor Bldg., Los Angeles, CA 90095-1689 (E-mail:
Ikurtz{at}mednet.UCLA.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.
Received 16 February 2001; accepted in final form 9 July 2001.
 |
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