Lens Epithelium and Fiber Na,K-ATPases: Distribution

and Localization by Immunocytochemistry

Margaret H. Garner and Yongli Kong

PURPOSE. To use immunofluorescence and immunogold techniques to identify the catalytic subunits
of the Na,K-ATPases of the lens and to determine their location in the cells of the epithelium and
cortex of bovine and human lenses.
METHODS. Frozen sections of capsulated and decapsulated bovine and human lenses were prepared,
blocked, and treated with affinity-purified polyclonal rabbit antibodies to the Na,K-ATPase catalytic
subunit isoforms with subsequent treatment with fluorescein isothiocyanate–labeled goat anti-
rabbit IgG and visualization of the fluorescence by light microscopy. An immunogold-labeled goat
anti-rabbit IgG was used to detect, by electron microscopy, the binding of the same affinity-purified
polyclonal antibodies to thin sections of decapsulated lenses that had been fixed and embedded in
Lowicryl K4M. The results were confirmed by staining of western blot analysis of sodium dodecyl
sulfate–polyacrylamide gel separations of enriched membrane preparations from bovine and hu-
man lenses.
RESULTS. The three common catalytic subunits of the Na,K-ATPases are present in the plasma
membranes of lens epithelium, lens fibers, or both. The data indicate a polarized distribution of the
a1 and a3 catalytic subunit isoforms in central epithelium. In the cortical fibers, the a2 isoform is
present around the interdigitations. The a3 isoform is found in the interdigitation-free regions of
human cortical fibers.
CONCLUSIONS. This unique distribution of Na,K-ATPases precludes the popular pump-leak model for
lens monovalent cation homeostasis. The functional significance of the distribution of Na,K-
ATPases in the lens epithelium and superficial fibers is currently under investigation. (Invest
Ophthalmol Vis Sci. 1999;40:2291–2298)

n many cataractous lenses, Na1 concentrations are ele-

I
vated and K1 concentrations are lower than normal.1,2 The
causes of the monovalent cation imbalances remain elu-
sive. The most popular theories involve increased plasma mem-
brane permeability with or without cation transporter inhibi-
tion. There is evidence that the Na,K-ATPases (monovalent
cation transporters) of the lens epithelium of cataractous hu-
man lenses are inhibited or that they display unusual steady-
state ATP hydrolysis kinetics.3,4 There is equally compelling
evidence to suggest that K1 transport is normal and that
membrane permeability is increased.5–7
Historically, the lens has been viewed as a syncytium8 –10
in which the lens epithelium is the site of Na,K-ATPase– de-
pendent Na1/K1 exchange. The lens fibers, on the other hand,
are believed to be permeable to Na1 and K1. This simple
model explains, quite adequately, the K1 current (anterior to
posterior) and the Na1 current (posterior to anterior) in nor-
mal lenses.11 More recently, data have been reported to sug-
gest that the lens fiber cells are rather impermeable and use
From the University of North Texas Health Science Center, De-
partment of Anatomy and Cell Biology, Fort Worth, Texas.
Supported by Grant EY07010 (MHG) from the National Eye Insti-
tute of the National Institutes of Health.
Submitted for publication January 21, 1999; revised April 4, 1999;
accepted April 28, 1999.
Proprietary interest category: N.
Corresponding author: Margaret H. Garner, Department of Anat-
omy and Cell Biology, University of North Texas Health Science Cen-
ter, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107.
their own Na,K-ATPases to maintain monovalent cation ho-
meostasis.12,13 Furthermore, there are two Na1 currents, the
one from anterior to posterior and one at the lens equator.14,15
The question then arises as to the mechanism by which
relatively impermeable fiber cells and the metabolically active
lens epithelium interact to maintain ion homeostasis. To an-
swer this question, the location and mechanism of action of the
major lens ion transporters and carriers need to be described
for normal clear lenses. Our focus has been the lens Na,K-
ATPases.12,13 In this report, we use immunocytochemistry to
describe the distribution of Na,K-ATPase catalytic subunits in
the plasma membranes of bovine lenses and clear human
lenses.
MATERIALS AND METHODS
Tissues
Bovine eyes, purchased from a local abattoir within 3 hours of
death, and human eyes, obtained within 12 hours of death
from the Fort Worth Eye Bank, were processed immediately.
Catalytic subunit-specific polyclonal peptide antisera, to
extracellular (EC) and intracellular (IC) epitopes of the three
catalytic subunit isoforms were prepared by injection of KLH-
peptide complexes into New Zealand white Rabbits. The pep-
tides used to make the antisera were MGKGVGRDKYEPAAVSE-
HGDKK for a1-IC, MGRGAGREYSPAATTAENGGGK for a2-IC,
MGDKKDDKSSPKK for a3-IC, GIRSATEEEPPNDDLYK for a1-
EC, GIKAAMEDEPSNDNLYK for a2-EC, and GIQAGTEDDPS-

Investigative Ophthalmology & Visual Science, September 1999, Vol. 40, No. 10
Copyright © Association for Research in Vision and Ophthalmology 2291

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 2292 Garner and Kong
GDNLYK for a3-EC. The peptides were prepared with an
Applied Biosystems Peptide Synthesizer at the UCLA Molecular
Biology Institute Peptide Synthesis Facility (model 430A). The
peptide sequences were confirmed by solid-phase sequence
analysis (Commonwealth Biotechnologies, Richmond, VA).
The resultant antisera were affinity purified. For the prep-
aration of each affinity resin, 4 ml of Affigel-Hz was reacted for
2 hours with 10 ml of 12.5% glutaraldehyde in 13 Affigel-Hz
coupling buffer (supplied by Bio-Rad, Hercules, CA). After the
coupling of glutaraldehyde, the resin was washed 2 times with
deionized water and 5 times with 100 mM phosphate buffer
(pH 6.0). Ten milliliters of peptide solution (0.5 mg/mL of the
specific peptide in 100 mM phosphate buffer, pH 6.0) and 2
mg of NaCNBH3 were added to the washed resin and mixed
overnight. The phosphate buffer was removed; the resin was
washed with 50 ml of 100 mM NaHCO3 and reacted for 30
minutes with an additional 3 mg of NaCNBH3 in the sodium
bicarbonate buffer. The NaCNBH3 was required to reduce the
Schiff base formed between the aldehyde of the resin and the
amine of the peptide or peptides. The resin was rinsed with
100 mM Na2CO3 (20 ml) followed by 50 ml of deionized water.
The resin was stored in 0.5 mM NaN3.
For affinity purification, the affinity resins were transferred
to Poly-Prep columns (Bio-Rad) and rinsed with 0.02 M Tris
buffer (TBST), pH 7.5, containing 0.150 M NaCl and 0.15%
Tween-20 (Bio-Rad). The resin (approximately 2 ml, packed)
was mixed with antiserum (0.5 ml diluted with 1.5 ml of TBST)
and placed on a shaker for 2 hours. The column was then
rinsed with 20 ml of TBST. Each TBST eluate was saved. Each
resin was then incubated for 1 minute in 0.1 M glycine (Gly)
buffer, pH 2.8. Each Gly eluate was collected in tubes contain-
ing 41 ml of 1 M Tris buffer, pH 9.5. Each resin was incubated
for 1 minute with an additional 1 ml of Gly buffer. These
eluates were collected in tubes containing 41 ml of 1 M Tris
buffer and combined with the previous Gly eluates. Sufficient
bovine serum albumin and NaN3 were added to the TBST and
Gly eluates to yield a final concentration of 1% and 0.1 mM,
respectively. The TBST and Gly eluents were aliquoted and
stored at 270°C.
The effectiveness of the affinity purification was tested by
slot blot analysis using rat brain microsomes (1 mg/mL) as the
antigen. For the slot blot analyses, the immune complexes
were visualized using horseradish-peroxidase–labeled goat an-
ti-rabbit IgG (hrp-GARIgG; Boehringer–Mannheim, Indianapo-
lis, IN) and the hrp-substrate, 4-chloro-1-napthol.
To test the specificity of the affinity purified antibodies,
western blot analysis of sodium dodecyl sulfate–polyacrylam-
ide gel electrophoresis (SDS–PAGE) separations of microsomal
fractions from rat brain, rat heart, and rat kidney were stained
with each of the six antibodies. Microsomal fractions were
prepared using previously described protocols.4,12,13,16 –21 Vi-
sualization was with hrp-GARIgG with 4-chloro-1-napthol as
the hrp-substrate. Interspecies cross-reactivity was checked
using rat, canine, and bovine microsomal preparations.
Immunofluorescence staining was performed on frozen
sections. Bovine and human lenses were cut into quarters and
fixed with 4% paraformaldehyde. After sequential immersion in
10%, 15%, and 20% sucrose solutions, the lens quarters were
embedded with OCT compound in liquid nitrogen. Cryosec-
tions at 6 mm, prepared with a cryostat, were collected on glass
slides and stained. Before staining, the sections were blocked
with normal goat serum for 1 hour before incubation with the
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IOVS, September 1999, Vol. 40, No. 10
primary antisera, followed by incubation with fluorescein iso-
thiocyanate–labeled GARIgG (FITC-GARIgG). For the negative
controls, sections were incubated with normal rabbit serum or
the appropriate TBST eluate, followed by incubation with
FITC-GARIgG. A Nikon Diaphot light microscope (403 objec-
tive) was used to visualize the stained sections. Photomicro-
graphs were collected as permanent records of the immuno-
fluorescence results. Exposure times of 15 seconds were used
for preparations treated with the affinity-purified antisera (Gly
fractions). Exposure times of 30 seconds were used for the
samples treated with the TBST fractions from the affinity puri-
fication of the antisera and for the normal rabbit serum con-
trols.
Immunoelectronmicroscopy was performed on isolated
lens fiber plasma membrane preparations as well as on fixed
bovine and human lenses. To prepare fractions enriched in
fiber cell plasma membranes from bovine and human lenses,
the following protocol was used. After the lens capsule epithe-
lium was removed, the lens cortex was collected as described
previously.12,13,21 The cortex was homogenized in ice-cold 0.5
M Tris buffer, pH 8.0. The homogenate was centrifuged at
7000 rpm for 15 minutes. The supernatant of the 7000 rpm
centrifugation was centrifuged at 25,000 rpm (Beckman Ti45
rotor) for 20 minutes. The resultant pellet was washed twice
with ice-cold 0.5 M Tris buffer. A microsomal fraction from the
bovine renal medulla was prepared using previously described
procedures.17 The lens and renal membrane preparations were
fixed (4% paraformaldehyde/0.1% glutaraldehyde) for 1 hour.
The fixed membranes were embedded in Lowicryl K4M. Thin
sections at 0.1 mm, on grids, were blocked for 1 hour in 50 mM
Tris buffer, pH 7.5, containing 0.05% Triton X-100 and bovine
serum albumin (5 mg/ml). The grids were incubated overnight
at 4°C with the appropriate primary antisera, rinsed, and incu-
bated with GARIgG coupled to 15 nm gold particles for 1 hour.
After rinsing, the sections were post-fixed with 2% glutaralde-
hyde and stained with uranyl acetate and lead citrate. For the
studies with the human lens, the quartered lens was fixed in 4%
paraformaldehyde/0.1% glutaraldehyde and embedded in Lowi-
cryl K4M. After removal of the nucleus from bovine lens
quarters, the remaining cortex was fixed in 4% paraformalde-
hyde/0.1% glutaraldehyde and embedded in Lowicryl K4M.
The procedure for preparation of and staining of the thin
sections on grids was identical with that described previously
for the lens fiber cell membranes. A Zeiss 910 transmission
electron microscope was used to visualize the stained sections.
Variation in immunogold-labeling patterns was deter-
mined from counts of gold particles in multiple fields. For
bovine lens fiber cell membrane preparations, gold particles
were counted in 48 fields (4 mm2 each) on 12 grids from two
separate membrane preparations. For human lens fiber cell
membranes, gold particles were counted in 35 fields (4 mm2
each) on seven grids from a single lens fiber cell membrane
preparation. The results for the membrane preparations are
reported as particles/4 mm2 field. For the bovine and human
lens sections, the total length of the membrane in 24 fields (4
mm2 each) was measured. The measured length of the mem-
brane in interdigitations was subtracted from the total length of
the membrane to determine the length of membrane devoid of
interdigitations. Counted gold particles in the membrane are
reported as particles per micrometer of membrane. The data
are presented as mean 6 SD. Using an unpaired Student’s t-test
 IOVS, September 1999, Vol. 40, No. 10 Immunocytochemical Localization of Lens Na,K-ATPases 2293

FIGURE 1. Antibody specificity was determined by staining of western blots of SDS–PAGE separations of
rat kidney (K), rat heart (H), and rat brain (B) microsomes with the affinity-purified antibodies (glycine
fraction) to the IC and EC epitopes of the a1, a2, and a3 catalytic subunit isoforms of the Na,K-ATPase.
SDS–PAGE separations were on 7% gels with 19 6 2 mg of the appropriate microsomal fraction per lane.
Primary antibody dilution was 1:10. Secondary antibody dilution was 1:1000. Between the IC and EC
panels is a representative standard for reference; standard molecular weights are (from top to bottom),
133, 115 kda (faint), 79, and 49 kDa.

(Statveiw; Abbacus), the significant differences in immunogold
labeling were determined.
RESULTS
Affinity Purification of the Six Antibodies
All three catalytic subunit isoforms are present in rat brain
microsomes. The Gly fractions of the affinity purification of the
six antisera stained rat brain microsome preparations on slot
blots. There was no staining of slot blots of rat brain micro-
some preparations with the TBST fractions. For all succeeding
experiments, the TBST fractions as well as normal rabbit serum
were used as negative controls for Gly fractions, used for the
western blot analysis and immunocytochemistry.
Specificity of the Six Antisera
Western blot analysis of SDS–PAGE separations of brain, kid-
ney, and heart microsome preparations was used to determine
the specificity of the affinity-purified antisera. Rat, canine, and
bovine microsome preparations were tested. The results for
the rat preparations (Fig. 1) are representative of these studies.
The data for the canine and bovine preparations are not in-
cluded and will be presented in a future report of our studies
with cultured lens epithelium.
The affinity-purified antisera a1IC and a1EC stained major
bands at ;95 kDa in all microsome preparations. This is in
agreement with previous studies that have demonstrated the
presence of the a1 isoform in kidney, heart, and brain. Lower-
molecular-weight bands are also stained with the a1IC and
a1EC antisera.22–26 The apparent molecular weights of the
lower-molecular-weight bands stained by the a1IC antiserum
are 70 kDa (brain), 40 to 42 kDa (heart and kidney), and 35 to
37 kDa (heart and kidney). Of these, only the 35- to 37-kDa
band is recognized with the a1EC antiserum. This band is
probably a cleavage product of the a1 subunit. Previous stud-
ies have demonstrated that the a1 isoform is highly susceptible
to proteolytic cleavage.27–30 The larger bands (70 kDa and the
40 – 42 kDa) are not stained with the a1EC antiserum but are
stained with the a1IC antiserum. The a1IC site is before the
a1EC site, and both EC and IC sites are within the first 250
residues. Therefore, the 70-kDa and 40- to 42-kDa bands are not
Na,K-ATPase degradation products but proteins that contain
regions homologous to the sequence chosen for the a1IC
antiserum.
The a2IC and a2EC antisera stained prominent bands at
95 kDa in the heart and brain preparations. There were no
bands stained in the kidney preparations. These results are in
agreement with previous studies that demonstrated the ab-
sence of a2 in the kidney and the presence of a2 in the brain
and heart.22,23,25,26,31 There was faint staining of a 70-kDa band
in the brain preparation by both the a2IC and a2EC antisera.
This suggests limited proteolytic cleavage.
The a3IC and a3EC antisera stained a 95-kDa band in
the brain preparation. There was no staining evident in the
kidney and heart preparations. These results are in agree-
ment with those of previous studies that have demonstrated
that the a3 isoform is found primarily in the central nervous
system.22,23,25,26,31
Localization by Indirect Immunofluorescence
The six antisera were used to determine the presence and
location of the Na,K-ATPase catalytic subunits in the bovine
lens by indirect immunofluorescence. The results for the three
EC antisera (Fig. 2) suggest that all three catalytic subunits are
present in the superficial regions of the bovine lens. The a1
isoform is present in the epithelium and in the superficial
fibers. In the central region of the lens epithelium (Fig. 2A) the
staining is heaviest on the basal and lateral surfaces. In the
equatorial epithelium (Fig. 2E) staining of the a1 isoform is
observed on the apical, basal, and lateral cell borders. In the
superficial cortical fibers, the a1 isoform is observed as punc-
tate spots on the fiber surface. There is little if any evidence of
the a2 isoform in the central epithelium because the fluores-
cence intensity shown in Figure 2B is similar to that for the
control (Fig. 2D). There is heavy staining of the equatorial
epithelium and superficial fibers with the a2EC antiserum. The
a2 isoform is observed as punctate spots on the surface of the
superficial fibers (Fig. 2F). The a3 isoform would appear to be
localized to the apical and lateral borders of cells in the central
epithelium (Fig. 2C). At the equator, the a3 isoform is present
on apical, basal, and lateral surfaces of the epithelium and on
the borders of the underlying fiber cells (Fig. 2G). Staining of
the lens epithelium and superficial cortical fibers was not
observed with the three IC antisera (data not included).

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 2294 Garner and Kong
FIGURE 2. Localization of Na,K-ATPase catalytic subunit isoforms of
the bovine lens epithelium. Indirect immunofluorescence with the
a1EC (A and E), a2EC (B and F), and a3EC (C and G) antisera and
sections from the central epithelium (A through D) and sections from
the equatorial epithelium with underlying superficial fibers (E through
H). (D and H) Representative controls with normal rabbit serum and
with the one of the TBST fractions (a2EC) from the affinity purifica-
tion, respectively. Controls with the TBST fractions from the affinity
purification of a1EC and a3EC were similar to (H), data not included.
The results with the three EC antisera (Fig. 3) suggest that
the catalytic subunits appear as patches on the fiber edge or in
punctate patterns on the fiber cell surface in the cortex of
decapsulated bovine and human lenses. In the deeper cortical
fibers of the bovine lens (Figs. 3A, 3D), staining with antisera to
the a2 and a1 isoforms is observed. There is little if any
evidence of staining with a3EC (Fig. 3G) in this region of the
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IOVS, September 1999, Vol. 40, No. 10
bovine lens. In the deeper fibers of the human lens (Figs. 3B,
3H), a2EC and a3EC stained the fibers. There is little if any
evidence of staining with a1EC (Fig. 3B) in this region of the
bovine lens. There was no evidence of staining with the three
IC antisera in this region of the lens (data not included).
Immunogold Labeling and Transmission Electron
Microscopy
To better characterize the discontinuous localization of the a2
and a3 catalytic subunit isoforms in human fiber cells, sections
from the superficial cortex of decapsulated human lenses were
immunogold-labeled and studied by electron microscopy. Al-
though the description of the immunogold-labeling results fo-
cuses on the quantitation of gold particles in multiple fields,
representative micrographs for the a1EC, a2 EC, and a3EC
antisera are shown in Figures 4A, 4B, and 4C, respectively.
There were 2 6 3 gold particles/mmmembrane for all the TBST
controls and for the a1EC antiserum. With the a2EC antiserum,
there were 46 6 16 particles/mmmembrane (P , 0.001 when
compared with the TBST control) adjacent to or associated
with interdigitations (see Fig. 4B as an example). In regions
devoid of interdigitations, the number of gold particles was
considerably lower (4 6 3 particles/mmmembrane) and not sig-
nificantly higher, statistically, than that of the controls (3 6 2
particles/mmmembrane; Figs. 4E and 4F). For the a3EC anti-
serum, gold particles were not prevalent in the interdigitations
(2 6 2 particles/mmmembrane) but were present in regions
devoid of interdigitations (10 6 5 particles/mm; P , 0.001).
Similar experiments carried out with the superficial cortex of
decapsulated bovine lenses suggested a2 at the interdigitations
(25 6 12 particles/mmmembrane; P , 0.001), a1 in the regions
relatively free of interdigitations (5 6 2 particles/mmmembrane;
P 5 0.048), and the absence of a3 (2 6 2 particles/mmmem-
brane; P . 0.1). Representative micrographs for the superficial
bovine lens cortex have not been included.
FIGURE 3. Localization of the Na,K-
ATPase catalytic subunit isoforms of
the cortex of decapsulated bovine
(A, C, D, G) and human (B, E, F, H,
I) lenses stained with a1EC (A
through C), a2EC (D through F), or
a3EC (G through I). (C, F, and I)
Representative of stains with the
TBST fractions from the affinity puri-
fication of a1EC, a2EC, and a3EC,
respectively. Magnification, 3100.
 IOVS, September 1999, Vol. 40, No. 10 Immunocytochemical Localization of Lens Na,K-ATPases 2295

FIGURE 4. Representative results for

immunogold labeling of sections
from the cortex of a human lens with
a1EC (A), a2EC (B), and a3EC (C
and D). (E) Normal rabbit serum con-
trol; (F) representative control with
a TBST fraction from the affinity
purification (a2EC). Magnification,
350,000.

The positive results were with the antisera to the extra- from the central epithelium of the bovine lens, a 90-kDa band
cellular epitopes. As pointed out in the previous paragraphs, was identified by the a1IC (Fig. 6, lane e) and the a3IC (Fig. 6,
similar studies with the antisera to the intracellular epitopes lane k) antisera; no bands were identified with the a2IC anti-
were negative. The lack of success with the three IC antisera serum (Fig. 6, lane h). For the membranes (a microsomal
was most likely a problem with penetration of the antiserum, fraction) from the equatorial epithelium of the bovine lens, a
availability of the antigenic determinant(s) in the fixed tissue, 90-kDa band was identified by the a1IC (Fig. 6, lane f) and a2IC
or both. To circumvent this problem, membranes, isolated (Fig. 6, lane i) antisera; lower-molecular-weight bands were
from the bovine renal medulla as well as bovine and human identified by the a1IC and a3IC antisera (Fig. 6, lanes f and l).
lens cortex, were fixed, immunogold-labeled, and studied by The a2IC antiserum identified a 90- to 95-kDa band from the
transmission electron microscopy. Representative micrographs SDS–PAGE separation of the water-insoluble fraction32 of a
for the a2IC and a2EC are shown in Figure 5. The quantitative decapsulated human lens. The human lens was obtained
results for multiple fields were collected into Table 1. Usually, within 6 hours of death. Studies with other human lenses
at least one gold particle appeared in all fields studied. There indicated significant degradation of the Na,K-ATPase catalytic
were no statistically significant differences among the values subunit of the lens fiber cells to molecular weight $10 kDa
for the TBST controls for the three membrane preparations. when lenses were received at times $10 hours postmortem.
The values for the a1IC and a1EC Gly fractions for microsomes Furthermore, with the significant loss of cells from the capsule
of the bovine renal medulla were significantly greater than epithelium of the postmortem human lenses, immunocyto-
those of the TBST controls, a result expected based on the chemical and immunoblot analysis results for the distribution
immunoblot data (Fig. 1). The values for the Gly fractions for of the Na,K-ATPase catalytic subunits were mixed and, there-
the a2 and a3 epitopes were not significantly different from fore, have not been included in this report.
those of the TBST controls. For the human lens membrane
preparations, the number of gold particles observed with the
Gly fractions of the antisera to the a2 and a3 epitopes was DISCUSSION
significantly greater than those of their respective TBST con- The results with the IC panel of antisera and the denatured lens
trols. Furthermore, the number of gold particles with the a2IC preparations suggest that a1 and a3 are the predominant
and a2EC Gly fractions was significantly greater than the num-
ber observed with the a3IC and a3EC Gly fractions. For the
bovine lens preparations, only the preparations stained with
the a2IC and a2EC Gly fractions had significantly more gold
particles than the respective TBST controls. This result was
unexpected because the results with a1EC antiserum in the
superficial bovine lens cortex suggested low levels of a1 in the
membrane regions free of interdigitations.
To confirm the immunocytochemistry, enriched mem-
brane fractions, isolated from the bovine lens central epithe-
lium, equatorial epithelium, cortex, and the decapsulated hu-
man lens were separated by SDS–PAGE, transferred to
nitrocellulose, and treated with the antisera to the catalytic
subunit isoforms (Fig. 6). For the membranes from the bovine
lens cortex, no bands were identified by the a1EC, a1IC, a3EC,
or a3IC antisera (data not included). With the a2IC antiserum
(Fig. 6, lane c) a band, MW ;90 kDa, was identified. The
90-kDa band and a 78-kDa band were identified with the a2EC FIGURE 5. Immunogold staining of membranes isolated from the bo-
antiserum (Fig. 6, lane d). This suggests cleavage of a 10- to vine lens cortex with the affinity-purified a2IC antiserum (A), the
12-kDa fragment from the amino terminus because the 78-kDa affinity-purified a2EC antiserum (B), the TBST fraction from the affinity
band was not apparent when the blot was stained with the purification of a2IC (C), and the TBST fraction from the affinity puri-
a2IC antiserum. For the membranes (a microsomal fraction) fication of a2EC (D). Magnification, 380,000.

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 2296 Garner and Kong IOVS, September 1999, Vol. 40, No. 10

TABLE 1. Immunogold Labeling of Isolated Membrane Fractions

Bovine Renal Medulla* Human Lens Fiber Cell* Bovine Lens Fiber Cell*
Microsomes Membranes Membranes

TBST Fraction Gly Fraction TBST Fraction Gly Fraction TBST Fraction Gly Fraction
Particles/ Particles/ Particles/ Particles/ Particles/ Particles/
Antiserum 4 mm2 4 mm2 4 mm2 4 mm2 4 mm2 4 mm2

a1IC 563 21 6 5† 363 464 564 566

a1EC 565 28 6 10† 463 563 565 664
a2IC 363 562 562 77 6 23† 563 23 6 9†
a2EC 465 461 564 62 6 30† 566 28 6 10†
a3IC 463 663 564 28 6 7†‡ 465 563
a3EC 263 563 465 24 6 10†‡ 465 865

EC, extracellular; Gly, glycine; IC, intracellular; TBST, Tris buffer with NaCl and Tween.
* Number of fields per antiserum fraction were 20, 35, and 48 for the renal medulla microsomes, human lens fiber cell membranes and bovine
lens fibercell membranes, respectively.
† Values significantly different between Gly and TBST fractions (the controls); P # 0.001.
‡ Comparisons of results with glycine fractions a2IC to a3IC and a2EC to a3EC in the human lens membrane preparations, P # 0.001.

isoforms in the bovine lens epithelium and that a2 is the
predominant form in the bovine and human lens fibers. This
result was confirmed by immunocytochemistry with the EC
panel. Immunocytochemistry results with the IC panel were
negative. Differential antibody penetration is a plausible expla-
nation for the disparate immunofluorescence results with the
two antibody panels. The epitopes, recognized by the three EC
antisera are extracellular.33–35 The epitopes for the IC antisera,
on the other hand, are intracellular.33 Permeabilization of the
lens sections with detergents or methanol, which was not part
of our experimental protocol, might have allowed adequate
antibody penetration to the site recognized by the IC antisera.
However, such treatments would be expected to compromise
the plasma membrane.
Even if permeabilization was not an issue, negative results
might be expected with the three IC antisera. The EC domain
of Na,K-ATPase catalytic subunits, proposed from analysis of
hydropathy plots and predicted secondary structure, consists
of relatively short segments of polar and/or charged amino
acids.36,37 It would be difficult to envision that these short
segments are so buried in the tertiary structure that they are
inaccessible to solvent, solutes, and antibodies. On the other
hand, the IC domain of the Na,K-ATPase catalytic subunits
consists of several long segments of sequence, the N-terminal
(;90 amino acid residues),33,38,39 the region between mem-
brane spanning sequences 2 and 3 (;125 amino acid resi-
dues),33 and the large region between membrane spanning
sequences 4 and 5 (;480 –500 amino acid residues).40 – 42 The
hydrophilic N-terminal epitopes, targeted by the three IC anti-
sera, may be buried in the tertiary structure, the quarternary
structure, or both of the native enzyme, thus unreactive. Alter-
natively, the antigenic determinants (epitopes) may be struc-
turally altered due to kinase-dependent phosphoryla-
tion38,43– 45 or inaccessible due to the interacting
cytoskeleton.42,46 – 48 Although future studies will address the
accessibility of the three IC epitopes in the native structure,
the results shown in Figures 1 and 6 clearly demonstrate
positive staining of a 90-kDa band by all six antisera when the
catalytic subunits are denatured and separated by SDS–PAGE.
To date, the only reported differences in the mechanism
of action of Na,K-ATPases containing the a1, a2, or a3 catalytic
subunit isoforms are the IC50 for Na1 inhibition of ATP hydro-
lysis (1.15 6 0.13, 1.05 6 0.11, and 3.08 6 0.06 mM, respec-
tively, for a1, a2, and a3) and the relative affinities for ATP
(0.43 6 0.12, 0.54 6 0.15, and 0.21 6 0.04 mM, respectively,
for a1, a2, and a3).34 A second question arose from this
observation, and a review of the results is presented in this
report. What is the purpose of multiple Na,K-ATPases in the
same cell?
In the central epithelium of the bovine lens, both a1 and
a3 appear to be expressed and to be localized in the plasma
membrane with the a1 basal and lateral and the a3 apical and
lateral. Chondrocytes of the articular cartilage and neurons are
the only other cells for which immunocytochemistry studies
have demonstrated the presence of the a1 and a3 catalytic
subunit isoforms in the same cell.24,49 Chondrocytes exist in a
unique environment in which free EC Na1 levels are 250 to
400 mM because of the high density of fixed negative charges
on the glycosaminoglycans in the EC matrix.49 The lens cap-
sule has a high glycosaminoglycan content, which may con-
tribute to elevated EC Na1 levels at the apical surface of the
lens epithelium. Whether elevated EC Na1 induces a3 catalytic
subunit expression remains to be determined. Similar to the
results with the cells of the central epithelium, the expression
of the a1 and a3 isoforms is polarized in neurons with the a1
predominant in the dendrite and a3 predominant in the ax-
on.24 It should be pointed out that the EC matrix of the central
nervous system is extensive and complex.50 Glycosaminogly-
cans are involved in central nervous system development and
have been implicated as players in the etiology of Alzheimer’s
disease.51–54 In fact, the adhesion molecule on glia (AMOG)
protein, a cell surface component involved in EC matrix inter-
actions in the brain, is one of the three possible glycoprotein
subunit isoforms of the Na,K-ATPase.55–58 Studies currently in
progress will address and clarify the issue of EC matrix inter-
actions with the Na,K-ATPases of the cells of the central epi-
thelium of the bovine lens. Other studies currently in progress
in our laboratory will address the role of the cytoskeleton in
the polarization of the a1 and a3 catalytic subunits in the
central epithelium of the bovine lens.
In the equatorial epithelium, there is an absence of a3/a1
polarization and the appearance of the a2 isoform, a result that
confirms previous studies of rat lens epithelium.13,59 Although
the importance of the changes in catalytic subunit distribution

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 IOVS, September 1999, Vol. 40, No. 10 Immunocytochemical Localization of Lens Na,K-ATPases
FIGURE 6. Immunostaining of western blot analysis confirms the re-
sults of the immunocytochemistry studies. Membranes isolated from
the cortex of decapsulated bovine lenses were separated by SDS–PAGE
on 7% gels (lanes b through d, 30 mg protein/lane) with the prestained
broad range SDS–PAGE standard (lane a) as a reference. Lane b is the
result of staining of the gel with Coomassie blue; lane c is the result of
staining of part of the western blot with a2IC; and lane d is the result
of staining of portion of the western blot with a2EC. Microsomal
fractions, isolated by differential centrifugation from the central epi-
thelium and equatorial epithelium of bovine lenses, were separated by
SDS–PAGE on 10% gels and transferred to nitrocellulose. Lanes e, h,
and k show the results of staining of the sections of the western blot
of the microsomal preparation of the central epithelium (20 mg pro-
tein/lane) with a1IC, a2IC, and a3IC, respectively. Lanes f, i, and l
show the results of staining of sections of the western blot of the
microsomal preparation of the equatorial epithelium (15–20 mg pro-
tein/lane) with a1IC, a2IC, and a3IC, respectively. Lanes g, j, and m
are the separated prestained low-range SDS–PAGE standard (Bio-Rad),
included on each gel as a reference. A portion (50 mg protein) of the
water insoluble fraction, isolated from a decapsulated human lens, was
separated by SDS–PAGE on a 10% to 20% gradient gel and transferred
to nitrocellulose. The blot was stained with the a2IC antiserum (lane
n); the prestained high-range molecular-weight standard, run with the
sample (lane o) as a reference.
in fibrogenesis remains to be elucidated, increased IC Na1
would appear to be the signal for induction of the a2 subunit.
Both an amphotericin-induced increase in membrane perme-
ability and cardiac glycoside inhibition of Na,K-ATPase have
been shown to induce the expression of the a2 subunit iso-
form.60,61
In the cortical lens fibers of bovine and human lenses, a2
is predominant. The a2 is a constituent of, or adjacent to,
interdigitations between cells. The a3 (human) isoform and
perhaps the a1 (bovine) isoform, which are present in much
lower concentrations than the a2 isoform, are restricted to
random patches in the interdigitation-free regions of the corti-
cal fiber membrane. Neither the a1 nor a3 isoforms is ob-
served, intact, on western blot analysis of membrane prepara-
tions from decapsulated bovine or human lenses. This would
suggest degradation of these isoforms in the membrane of the
fully differentiated cortical fiber.
It is obvious that the regulation of monovalent cation
homeostasis is rather complex in this syncytium, the lens. The
importance of the active transport of 86Rb1 by the fibercell
Na,K-ATPase has already been established for the bovine lens
in organ culture.12 The role of the polarized distribution of two
distinct Na,K-ATPases in the membrane of the cells of the
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2297
central epithelium remains to be defined. The importance of
the subsequent degradation of the epithelial cell Na,K-ATPases
in mature fibers also remains to be elucidated. Finally, the
changes in monovalent cation homeostasis in humans with
cataract may or may not be the result of Na,K-ATPase inhibition
as currently believed. Changes in Na,K-ATPase distribution,
synthesis, and degradation could be expected to play a role as
well.
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