Na,K-ATPase Polypeptide Upregulation Responses in
Lens Epithelium
Nicholas A. Delamere,13 Robert E. Manning, Jr.,1 Lixia Liu,1 Amy E. Moseley,2 and
William L Dean*
In a previous study, an increase in Na,K-ATPase a2 expression was detected in the
epithelium of porcine lenses exposed to amphotericin B, an ionophore that also increases lens
sodium and stimulates active sodium transport. The purpose of the present study was to determine
whether an increase of Na,K-ATPase a2 synthesis is a response to an episode of rapid Na-K transport
or whether the increase in lens sodium alone can initiate the response.
PURPOSE.
Western blot analyses were conducted to probe for Na,K-ATPase a polypeptides in
membrane material isolated from porcine lens epithelium. Ouabain-sensitive adenosine triphosphate hydrolysis was used as an index of Na,K-ATPase activity, and lens ion content was determined
by atomic absorption spectrophotometry. 86-Rubidium (86Rb) uptake was measured as an indicator
for active potassium transport.
METHODS.
86
Rb uptake was markedly diminished in lenses exposed to dihydro-ouabain (DHO),
signifying inhibition of active sodium-potassium transport. Consistent with this, the sodium
content of DHO-treated lenses increased. By western blot analysis, a marked increase of Na,KATPase a2 polypeptide could be detected in the epithelium of DHO-treated lenses. To rule out the
possibility that apparent stimulation of Na,K-ATPase a2 synthesis stemmed from binding of DHO to
Na,K-ATPase sites, experiments were conducted to confirm an increase of Na,K-ATPase a 2 polypeptide in the epithelium of lenses exposed to low-potassium medium to inhibit active sodiumpotassium transport. Consistent with the apparent increase of Na,K-ATPase polypeptide, Na,KATPase activity was detectably increased in epithelial material isolated from lenses pretreated with
DHO or low-potassium medium.
RESULTS.
An increase in Na,K-ATPase a2 polypeptide can occur in the epithelium of lenses
subjected to an episode of sodium pump inhibition. This sviggests the response could be triggered
by an increase in cell sodium and does not necessarily require a period of stimulated active
sodium-potassium transport. {Invest Ophthalmol Vis Set 1998;39:763-768)
CONCLUSIONS.
A
ctive sodium-potassium (Na-K) transport, mediated by
sodium, potassium, and adenosine triphosphatase
(Na,K-ATPase), is vital for the maintenance of cytoplasmic ion composition in the lens. This is important because
when the lens sodium content is allowed to increase, there is
a concomitant decrease in potassium and an increase in calcium in the lens. These changes can lead to cell damage and
loss of lens transparency. It is noteworthy that most human
lenses with cortical opacification display marked disturbance
of cation content.1
The fiber cells, which make up the bulk of the lens,
have low specific Na,K-ATPase activity, and it is widely
thought that the anterior monolayer of epithelial cells plays a
From the Departments of 'Ophthalmology and Visual Sciences,
Biochemistry and 3Pharmacology and Toxicology, University of Louisville, Kentucky.
Supported by United Health Public Services research grant
EY09532 and the Kentucky Lions Eye Foundation, and by an unrestricted grant from Research to Prevent Blindness, New York.
Submitted for publication July 7, 1997; revised December 2, 1997;
accepted December 18, 1997.
Proprietary interest category: P.
Reprint requests: Nicholas A. Delamere, Department of Ophthalmology and Visual Science, University of Louisville School of Medicine,
301 East Muhammad Ali Boulevard, Louisville, KY 40292.
2
key role in conducting active Na-K transport, which underpins cation homeostasis for the entire lens cell mass. The
lens epithelium has high specific activity of Na,K-ATPase,
presumably giving the cells a large capacity for active Na-K
transport. However, it is known that the lens becomes increasingly permeable with age.2 In an earlier study, we
tested whether the epithelium can increases Na,K-ATPase activity to meet the demands of cation homeostasis in a lens in
which membranes are leaky. In porcine lenses made permeable with the pseudoionophore amphotericin B, evidence
suggests that the epithelium can increase the abundance of
Na,K-ATPase protein.3 This response appears to involve stimulation of the biosynthesis of the Na,K-ATPase a2 isoform.
We wanted to determine the cellular mechanisms that trigger Na,K-ATPase a2 synthesis. Knowing that amphotericin B
causes an immediate stimulation of the sodium pump rate,
we speculated that Na,K-ATPase a2 synthesis could be a response to an episode of rapid Na-K transport. However, in
the present study, we present evidence suggesting that stimulation of the sodium pump may not be necessary for stimulation of Na,K-ATPase a2 biosynthesis to occur. The results
show that Na,K-ATPase a2 polypeptide abundance increases
in the epithelium of porcine lenses exposed to dihydroouabain (DHO), which inhibits active Na-K transport.
Investigative Ophthalmology & Visual Science, April 1998, Vol. 39, No. 5
Copyright © Association for Research in Vision and Ophthalmology
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Delamere et al.
MATERIALS AND METHODS
Chemicals
Methylene-bisacrylamide, ammonium persulfate, N,N,N',N'-tetramethylethylenediamine and goat antirabbit horseradish peroxidase-conjugated antibodies were obtained from Bio-Rad
(Richmond, CA); reagents for detection of immunoblots by
chemiluminescence from DuPont NEN (Boston, MA); and
bicinchoninic acid protein assay reagent from Pierce Chemical
(Rockford, IL). Other chemicals were obtained from Sigma
Chemical (St. Louis, MO).
Lenses
The procedures used in these studies were approved by the
University of Louisville Institutional Animal Care and Use Committee and conformed with the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research. Porcine eyes were
obtained from a local abattoir. The posterior of the eye was
dissected, and the suspensory ligaments of the lens were cut
with fine scissors. The intact lens was gently removed and
incubated for specified times in serum-free Dulbecco's modified Eagle's medium (DMEM) (Sigma) supplemented with 100
U/ml penicillin and 100 /xg/ml streptomycin at 37°C in a
humidified atmosphere of 95%:5% air:CO2. The cation concentrations in the DMEM were 155 mM Na+, 5.3 mM K+, 1.4 mM
Ca2+, and 0.8 mM Mg2+. In some experiments, lenses were
incubated in low-potassium DMEM containing 0.5 mM potassium. To maintain osmolarity, an equimolar amount of NaCl
was added in replacement of KC1.
86-Rubidium Uptake
Ouabain-sensitive 86-rubidium (86Rb) uptake by the lens was
used as an index of active inward potassium transport mediated by Na,K-ATPase. It was assumed that 86Rb is transported
similarly to potassium. 86Rb uptake was determined in lenses
immersed in DMEM, in the presence or absence of DHO or in
low-potassium DMEM. Lenses were exposed to DHO or lowpotassium DMEM for 10 minutes, and approximately 0.1
/LtCi/ml 86Rb ± 1 mM ouabain was added for a further 60
minutes. During this time, 86Rb uptake was linear. After the
86
Rb uptake period, each lens was removed from the radioactive medium and was placed in a large volume of ice-cold
nonradioactive Krebs solution, which contained 119 mM NaCl,
4.7 mM KC1, 1.2 mM KH2PO4) 25 mM NaHCO3, 2.5 mM CaCl2,
1 mM MgCl2, and 55 mM i>glucose (pH 7.4) for 5 minutes to
wash out extracellular 86Rb. The lenses were blotted dry,
weighed, and digested in 30% nitric acid. 86Rb in the digest and
the loading medium was measured by scintillation counting.
The data were used to calculate the uptake of potassium by the
lens, presented as nanomoles per gram wet weight per hour.
Measurements of Lens Sodium and Potassium
Content
As described in an earlier article,3 lenses were weighed, dried,
and digested in 30% nitric acid and sodium, and potassium was
quantified using an atomic absorption spectrophotometer
(model 372; Perkin-Elmer, Norwalk, CT). The data are expressed as nanomoles per kilogram lens water.
Membrane Preparation and Western Blot Analysis
The capsule-epithelium was removed from each lens. The
epithelial monolayer is tightly attached to the capsule so that
IOVS, April 1998, Vol. 39, No. 5
when the capsule is removed, most of the epithelial cells but
few fiber cells remain attached to the capsule.4 As described
previously,3 the protein composition in membrane material
isolated from lens capsule- epithelium and from lens fiber cells
was compared to verify that capsule-epithelial preparations
were not significantly contaminated with fiber cells.
Membrane material was isolated from lens capsule-epithelium or from lens cortex (fiber cells). Tissue samples were
homogenized in ice-cold buffer A, which contained protease
inhibitors antipain (16.5 /xM), leupeptin (21 jLtM), pepstatin A
(14 piM), phenylmethylsulfonyl fluoride (40 /xM), and aprotinin
(0.027 trypsin inhibitor units per milliliter). The homogenate
was placed in a centrifuge for 60 minutes at 115,000,g. To
remove extrinsic protein, the pellet was resuspended in buffer
containing 600 mM KC1 and was subjected to centrifugation
for 60 minutes at 115,000g. The pellet was resuspended in
buffer A and was returned to the centrifuge for a final 60
minutes at 115,000g. Thefinalpellet of water-insoluble membrane material was resuspended in buffer A, divided into aliquots, and stored at —70°C. Protein content of the membrane
material was measured by bicinchoninic acid assay,5 using
bovine serum albumin as a standard. Membrane material isolated from porcine brain was used as a positive control for
Na,K-ATPase immunoblots. The isolation of brain membrane
material is detailed by Moseley et al.6
Proteins were separated by electrophoresis on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel, using the Laemmli7 buffer system. Molecular weight markers or membrane
protein material (60 jug) was applied to each lane. After electrophoresis, the separated proteins were electrophoretically
transferred to nitrocellulose sheets in transfer buffer that contained 25 mM Tris, 192 mM glycine, and 10% methyl alcohol
(pH 8.3). The nitrocellulose was blocked with 2% ovalbumin in
0.03 M Tris buffer (pH 7.5), 0.15M NaCl, and 0.5% Tween 20
(TTBS) for 1 hour and was incubated for a further hour with
the primary Na,K-ATPase antibody (rabbit anti-rat Na,K-ATPase). Polyclonal antibodies directed against the a, and a 3
isoforms of Na,K-ATPase were purchased from Upstate Biotechnology (Lake Placid, NY). The monoclonal antibody directed against the Na,K-ATPase a2 isoform, McB2, a gift from
Kathleen J. Sweadner, was raised against purified Na,K-ATPase
of rat brain axolemma.8 After incubation with primary antibody, the nitrocellulose was washed five times with TTBS and
was incubated for 1 hour with a horseradish peroxidaseconjugated secondary antibody. The nitrocellulose was washed
five times with TTBS, and the Na,K-ATPase polypeptide immunoblots were visualized by incubating the nitrocellulose with
chemiluminescence substrate for 1 minute and exposing the
sheet to radiographic film for 30 to 90 seconds.
Measurements of Na,K-ATPase Activity in Lens
Epithelium
Lenses were incubated for 24 hours in DMEM, containing
DHO, low-potassium DMEM, or DMEM alone (control), and
were incubated a further 3 hours in DMEM alone to wash out
the test solution. The capsule-epithelium was removed from
each lens and was homogenized in an ice-cold buffer containing 40 mM histidine, 100 mM NaCl, 10 mM KC1, 3 mM MgCl2
and 1 mM EGTA (pH 7.4). The mixture was divided into
aliquots, half of which received ouabain added to afinalconcentration of 1 mM. The samples were warmed to 37°C for 5
minutes, and adenosine triphosphate (ATP) was added to a
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Lens Na,K-ATPase
IOVS, April 1998, Vol. 39, No. 5
final concentration of 2.5 mM. After 30 minutes' incubation,
ice-cold trichloroacetic acid was added to terminate ATP hydrolysis. ATP hydrolysis was quantified by determining the
release of inorganic phosphate, using a colorimetric method
based on the work of Bonting et al.9 Protein was measured in
an aliquot of each homogenate, and ATPase activities were
calculated as nanomoles phosphate released per milligram protein. Na,K-ATPase activity was specified as the difference in
ATP hydrolysis measured in the presence and absence of
ouabain.
1bU
c
2c
"5
0
0
100
c
0
<D
O
50
Although the results are not quantitative, the results of the
immunoblot experiments suggest that Na,K-ATPase a2 isoform
polypeptide abundance is increased in the epithelium of lenses
*
I
In
1
[ II
•
153
3
INfl jj
i
j1
'21
1
CTRL DHO
Sodium
-9
B
1
jjug 9
(0 E
0 E
RESULTS
Lenses incubated 24 hours in the presence of 5 /xM DHO were
observed to have a significantly (P < 0.01) elevated sodium
content and reduced potassium content (Fig. 1A) consistent
with an episode of inhibited active Na-K transport. As judged
by 86Rb uptake studies, DHO inhibits ouabain-sensitive potassium (86Rb) uptake almost immediately. 86Rb uptake by intact
porcine lenses was measured in the presence of DHO added 10
minutes before the addition of 86Rb. At the 5- to 10-/xM test
concentrations used in this study, DHO inhibited more than
90% of ouabain-sensitive potassium (8<5Rb) uptake (Fig. IB).
This response is consistent with Na,K-ATPase inhibition: The
dose-response correlation for the inhibitory effect of DHO on
Na,K-ATPase activity in membrane material isolated from porcine lens capsule- epithelium or brain was similar to the doseresponse correlation for the inhibitory effect of DHO on 86Rb
uptake by the intact lens (Fig. IB).
In some studies, intact porcine lenses were incubated 24
hours in the presence of DHO, and membrane material was
isolated from the lens epithelium and examined by western
blot analysis, in which antibodies directed against the Na,KATPase a,, a2 and a 3 isoforms were used (Fig. 2). Antibody
specificity has been established previously by verification that
the antibodies reveal the expected patterns of Na,K-ATPase aL,
a-2, and a 3 expression in porcine brain, skeletal muscle, and
kidney medulla.3 In epithelial cell membrane material isolated
from fresh lenses or control incubated lenses, a dense band
was observed for Na,K-ATPase a, immunoreactive polypeptide, but Na,K-ATPase a 2 immunoreactive polypeptide was not
detected or appeared as a faint band. In plain contrast, a
marked Na,K-ATPase a2 immunoblot was observed together
with an a, immunoblot in epithelium membrane material isolated from lenses incubated 24 hours in the presence of DHO.
In view of the inevitable cell deterioration caused by Na,KATPase inhibition, it is not surprising that there was some
variability in a, and a2 immunoblot density among different
groups of DHO-treated lenses, hi all cases, however, DHO
treatment caused an increase of a2 polypeptide but no disappearance of a, polypeptide in the epithelium. Na,K-ATPase a3
immunoreactive polypeptide was not detected in control
lenses or in lenses incubated in the presence of 5 JU,M DHO
(data not shown). As described earlier,3 only the Na,K-ATPase
a, isoform is detectable in membrane material isolated from
control porcine lens fibers. Na,K-ATPase a2 immunoreactive
polypeptide was not detected by western blot analysis in fiber
material from control or DHO-treated lenses.
765
-7
1
-1
i
i 1
if
CTRL DHO
Potassium
-5
-3
Log DHO concentration (M)
1. (A) Sodium and potassium content of lenses incubated for 24 hours in the presence or absence (control) of 5
JJM dihydro-ouabain (DHO). The data are mean ± SE; n = 10
to 12 lenses. *Indicates a significant difference (P < 0.01) from
control values. (B) Dose-response correlation for the inhibitory effect of DHO on ouabain-sensitive 86Rb uptake by intact
porcine lens (•). Ouabain-sensitive potassium (86Rb) uptake
was measured in intact lenses incubated in the presence or
absence (control) of DHO, added 10 minutes before the start of
the 60-minute uptake period. The data are the mean of values
from two lenses at each DHO concentration. The control
uptake rate was 2080 ±210 nmol potassium (86Rb) per gram
wet weight per hour. (Mean ± SE; n = 6). For comparison, the
graph also shows the dose-response correlation for the inhibitory effect of DHO on Na,K-ATPase activity measured in membrane material isolated from porcine lens epithelium (A) and
porcine brain (•). Na,K-ATPase activity measurements were
made using pools of membrane material isolated from several
tissue samples. Data are the mean of four measurements at
each DHO concentration; SE values (less than 10%) have been
omitted for clarity.
FIGURE
subjected to prolonged inhibition of active Na-K transport by
DHO. To rule out the possibility that the responses were
caused by binding of DHO to Na,K-ATPase sites, some experiments were conducted in which active Na-K transport was
inhibited by exposing the lens to DMEM with a low-potassium
concentration (0.5 mM). Shortly after the lenses were placed in
low-potassium DMEM, ouabain-sensitive potassium (86Rb) uptake was reduced from a control value of 2011 ± 223 to 173 ±
35 nmol/g wet weight per hour (mean value ± SE; n = 8).
However, it should be noted that with time, potassium leaving
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766
Delamere et al.
IOVS, April 1998, Vol. 39, No. 5
* 116
- 85
1 2
3 4
t
116
85
B
12
3 4
FIGURE 2. Western blot analysis to determine Na.K-ATPase a,
.immunoreactive polypeptide (A) and a2 immunoreactive
polypeptide (B) in membrane material isolated from the epithelium of intact lenses incubated 24 hours in the presence of
5 /xM diliydro-ouabain (DHO) (lane 1), 10 JLIM DHO (lane 2) or
control lenses that did not receive DHO (lane 3). Membrane
material isolated from brain (lane 4) was used as a positive
control. (C) Western blot for Na,K-ATPase a2 immunoreactive
polypeptide in membrane material isolated from fiber cells
(cortex) of lenses incubated 24 hours in the presence of 5 /xM
DHO (lane 1) or 10 /xM DHO (lane 2). No immunoreactive
band was detected, although a dense band was observed in the
positive control (brain; lane 3). The Na,K-ATPase a, and a2
immunoreactive bands were detected at 100 to 105 kDa. The
position of molecular size markers is indicated by arrowheads
to the right of each blot.
water (mean ± SE; n = 10). Na,K-ATPase a2 responses observed in lenses exposed to low-potassium DMEM were similar
to the responses observed in lenses exposed to DHO. A dense
a2 immunoblot was observed in material isolated from lenses
exposed 24 hours to low-potassium DMEM (Fig. 3). Na,KATPase a, immunoblot density did not appear to diminish after
lens incubation in the presence of low-potassium DMEM. Na,KATPase a 3 polypeptide could not be detected.
The appearance of Na,K-ATPase a 2 polypeptide in the
epithelium of lenses exposed to DHO or low-potassium DMEM
suggests there could be a net increase of Na.K-ATPase protein.
To test whether this is paralleled by an increase of Na,K-ATPase
activity, intact porcine lenses were incubated 24 hours in the
presence of 5 JLLM DHO or in low-potassium DMEM. To wash
out the test solution, the lenses were cultured a further 3 hours
in three changes of control medium. After this, the epithelium
was removed from each lens and was used for measurements
of Na,K-ATPase activity (ouabain-sensitive ATP hydrolysis). Despite this, Na:K-ATPase activity in membrane material isolated
116
85
114
1 2 3
1
2
a 1
the lens will cause a slight increase in potassium concentration
in the bathing medium, which would lessen the degree of
sodium pump inhibition. Nevertheless, after incubation in lowpotassium DMEM for 24 hours, lens sodium content was increased from a control value of 20.7 ±1.4 mmol/kg lens water
to a value of 50.8 ± 5-3 mmol/kg lens water; lens potassium
content was decreased from a control value 132.6 ± 3.5
mmol/kg lens water to a value of 98.8 ± 3.8 mmol/kg lens
FIGURE 3-
*• 116
-85
1
2
a 2
Western blot analysis to determine Na,K-ATPase a,
immunoreactive polypeptide (left) and a2 immunoreactive
polypeptide (right) in epithelium membrane material isolated
from control lenses (lane 2) or lenses exposed to low-potassium medium 24 hours (lane 1). The Na,K-ATPase a, and a 2
immunoreactive bands were detected at 100 to 105 kDa. The
position of molecular size markers is indicated by arrowheads
to the right of each blot.
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Lens Na,K-ATPase
600
*-" o
0 CO
to
500
0)
400
QL
h
<
otei
min
IVlt
IOVS, April 1998, Vol. 39, No. 5
Q.
O)
E
300
200
2>
(0 E 100
z
0
CTRL
DHO
treated
Low K
treated
FIGURE 4.
Na,K-ATPase activity measure in epithelial material
isolated from intact lenses that had been incubated 24 hours in
the presence or absence (control) of 5 JLLM dihydro-ouabain
(DHO) or low-potassium (Low K) medium, and incubated 3
hours in three changes of control medium. The data are the
mean ± SE; n = 6 to 10 lenses. *Signines a significant difference (P < 0.05) from control values. The difference between
values in the dihydro-ouabain and low-potassium groups was
not significant.
from the epithelium of lenses that had been cultured 24 hours
in the presence of 5 /xM DHO was significantly higher (P <
0.05) than was the activity measured in membrane material
isolated from control lens epithelium (Fig. 4). The epithelial
Na,K-ATPase activity of lenses incubated 24 hours in lowpotassium DMEM was also significantly higher (P < 0.05) than
was the activity in control samples, but it was not significantly
different from the activity determined in the epithelium of
DHO-treated lenses. However, it should be made clear that
although DHO is a reversible Na,K-ATPase inhibitor,10 we did
not confirm that membrane material isolated from DHO-treated
lenses was completely free of residual DHO contamination;
considerable variability of Na,K-ATPase activity values was observed when DHO-treated lenses were washed briefly (5 minutes) in control medium, rather than subjected to a 3-hour
washout interval before the ATP hydrolysis measurement.
DISCUSSION
In an earlier study, we demonstrated that Na,K-ATPase a2
abundance is increased in the epithelium of lenses in which
the sodium pump rate has been stimulated for 24 hours by
exposure to the pseudoionophore amphotericin B.3 Here, we
show a similar Na,K-ATPase a 2 response in lenses exposed to
the Na,K-ATPase inhibitor DHO. Although Na,K-ATPase a2
polypeptide is difficult to detect by western blot analysis in
freshly isolated porcine lens epithelium, a marked Na,K-ATPase
a2 immunobtot was observed in epithelium samples obtained
from lenses incubated with DHO for 24 hours. Amphotericin B
and DHO have opposite effects on the sodium pump rate.
Amphotericin B increases the sodium pump rate secondary to
increasing sodium permeability, whereas DHO is a specific,
reversible, Na,K-ATPase inhibitor.10 The micromolar concentration range of DHO used in this porcine tissue study was
sufficient to inhibit more than 90% of Na,K-ATPase activity,
767
even though the only Na,K-ATPase isoform detected in the lens
of this species is a v 3 This differs from the situation in nit, in
which the a, isoform is relatively insensitive to ouabain8 and
freshly isolated lenses also express detectable amounts of the
a2 and a 3 Na,K-ATPase isoforms.6 To rule out the possibility
that the Na,K-ATPase a2 response stemmed from binding of
DHO to the Na,K-ATPase a subunit molecule, a second set of
experiments was conducted in which lens active Na-K transport was inhibited by reducing the external potassium concentration. A marked increase in Na,K-ATPase a2 polypeptide
abundance was also observed in the epithelium of these lenses.
The observed increase of Na,K-ATPase a 2 polypeptide abundance in the epithelium of lenses incubated in the presence of
DHO or in low-potassium medium suggests that the apparent
increase in the amount of a2 polypeptide does not require an
episode of stimulated sodium pump rate as a trigger.
Compared with the Na,K-ATPase activity determined in
the epithelium of lenses incubated under control conditions,
there was an increase of Na,K-ATPase activity in the epithelium
of lenses incubated 24 hours in the presence of DHO or
low-potassium medium. It is tempting to speculate that part of
the Na,K-ATPase activity increase stemmed from the increased
abundance of Na,K-ATPase a2 polypeptide in these lens epithelium samples. However, it is also possible that the observed
Na,K-ATPase activity increase could be the result of biochemical changes that alter the activity of the original pool of
Na,K-ATPase a, polypeptide.
In several cell types, it has been suggested that increased
cytoplasmic sodium levels can lead to Na,K-ATPase polypeptide
upregulation.1112 This notion agrees with the present observation that Na,K-ATPase a2 abundance is increased in the epithelium of DHO-treated lenses and lenses incubated in low-potassium
medium, both of which have a marked increase of sodium content. It also agrees with the observation of lens epithelium responses to amphotericin B, in that this increases lens sodium as
well.3 Indeed, it has been suggested by Yamamoto et al.13 that
Na,K-ATPase gene regulation could be linked to cytoplasmic sodium concentration. However, the possibility cannot be excluded
that changes in Na,K-ATPase a2 expression in the lens epithelium
are triggered by mechanisms involving alteration of cytoplasmic
calcium concentration, alteration of cytoplasmic pH, or other
cellular changes that could be associated with increased cytoplasmic sodium concentration.
Because the objective of the present study was to test
whether a sodium pump rate increase is required as a trigger for
Na,K-ATPase a2 response, DHO treatment was used simply as an
experimental maneuver to increase lens sodium content without
increasing the rate of active Na-K transport. However, there is
considerable evidence that endogenous ouabain-like molecules
have a local hormone-like action in modulating sodium pump
function.14 Considering the report that a ouabain-like molecule is
present in human cataractous lenses,15 there is a possibility that
lens Na,K-ATPase could be exposed to such inhibitory factors in
vivo. In the Nakano mouse, a strain daat is prone to cataract
development, there is persuasive evidence for the inliibition of
lens Na,K-ATPase by what seems to be an endogenous molecule.16 In addition, inhibition of the sodium pump has been
suggested as a stimulus for increasing Na,K-ATPase expression in
other tissues, in which it has been proposed that the response
could be initiated when reduced extracellular potassium concentration causes Na.K-ATPase inhibition.12
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Delamere et al.
During the 24-hour period of the present studies, exposure of the lens to DHO or low-potassium medium stimulated
the apparent synthesis of Na,K-ATPase a2 polypeptide in the
epithelium but not in fibers. The similarity of this pattern of
changes to those observed in lenses exposed to amphotericin
B suggests that the a2 isoform could be an inducible Na,KATPase isoform in porcine lens. However, it should be made
clear that on the basis of the present results, it cannot be
determined whether the increased a2 abundance represents an
upregulation involving changes in the abundance of a2 mRNA
or whether the response involves a change in polypeptide
turnover. In vivo, it is possible that regulation of a2 synthesis in
the epithelium serves to tailor the sodium pump capacity of
the lens to episodes of increased ion permeability. However, it
should be noted that although there is no evidence for changes
in Na,K-ATPase a, or a 3 polypeptide synthesis in lens epithelium, it is possible that such changes could occur during a
longer period or could be activated by different stimuli. It has
been reported that in cultured dog lens epithelium, doubling
osmolarity in the culture medium stimulates Na,K-ATPase a,
synthesis.17 The situation is made complex because between
different tissues, there is considerable variability in Na,K-ATPase responses. In the rat, for example, aldosterone causes
upregulation of the Na,K-ATPase a, isoform in cardiocytes18
but selectively increases Na,K-ATPase a5 isoform expression in
the hippocampus.19 Adding to the complexity, the pattern of
Na,K-ATPase isoforms expressed in the lens seems to differ
between species: Na,K-ATPase a2 isoform polypeptide is abundant in the epithelium of freshly isolated rat lens6 but not
porcine lens3 or bovine lens.20 Moreover, lens fiber cells express a considerable amount of Na,K-ATPase polypeptide, detectable by immunoblot2021 and ouabain binding.22 Results of
studies by Garner23 suggest a functional contribution of fiber
cell Na,K-ATPase to cation homeostasis in the bovine lens.
The lens faces unusual challenges because a relatively tiny
fraction of the cells remain as undifferentiated epithelial cells that
preserve high Na,K-ATPase activity. Moreover, lens cells are long
lived. Thus the ability of the epithelium to increase Na,K-ATPase
activity may be an important protective mechanism, enabling the
lens to cope with increased ion permeability, which appears to
occur as people age.2 Studies of transparent human lenses show
evidence for a detectable age-related increase of calcium ATPase
in the lens epithelium.24 It remains to be tested whether this is
also die case with Na,K-ATPase.
References
1. Duncan G, Bushel! AR. Ion analysis of human cataractous lenses.
Exp Eye Res. 1975;20:223-230.
2. Duncan G, Hightower R, Gandolfi A, Tomlinson J, Maraini G.
Human lens membrane cation permeability increases with age.
Invest Ophtbalmol Vis Sci. 198930:1855-1859.
IOVS, April 1998, Vol. 39, No. 5
3. Delamere NA, Dean WL, Stidam JM, Moseley AE. The influence of
amphotericin B on the sodium pump of porcine lens epithelium.
AmJ Physiol. 1996;270:C465-C473.
4. Alvarez LJ, Candia OA, Grillone LR. Na+-K+-ATPase distribution in
frog and bovine lenses. Curr Eye Res. 1985;4:l43-152.
5. Smith PK, Krohn RI, Hermanson GT, et al. Measurement of protein
using bicinchoninic acid. Anal Biochem. 1985;150:76-85.
6. Moseley AE, Dean WL, Delamere NA. Isoforms of Na,K-ATPase in
rat lens epithelium and fiber cells. Invest Ophthalmol Vis Sci.
1996;37:15O2-15O8.
7. Laemmli UK. Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nat Lond. 1970;217:680-685.
8. Urayama O, Shutt H, Sweadner KJ.Identification of three isozyme
proteins of the catalytic subunit of the Na,K-ATPase in rat brain.
JBiolChem. 1989;264:8271-8280.
9. Bonting SL, Simon KA, Hawkins NM. Studies on sodium-potassium-activated adenosine triphosphatase. I: quantitative distribution
in several tissues of the cat. Arch Biochem Biophys. 196l;95:4l6423.
10. Cox TC, Woods RE. Dihydroouabain a reversible inhibitor of the
sodium pump in frog skin. Eur Physiol. 1987;409:323-327.
11. Pressley TA. Ion concentration-dependent regulation of Na,Kpump abundance. J Membr Biol. 1988;105:187-195.
12. Wolitzky BA, Fambrough DM. Regulation of the (Na+-K+)-ATPase
in cultured chick skeletal muscle: modulation of expression by the
demand for ion transport. / Biol Chem. 1986;26l:9990-999913- Yamamoto K, Ikeda U, Seino Y, et al. Regulation of Na,K-adenosine
triphosphatase gene expression by sodium ions in cultured neonatal rat cardiocytes. / C/m Invest. 1993;92:1889 -1907.
14. Blaustein MP. Endogenous ouabain: role in the pathogenesis of
hypertension. Kidney Int. 1996;49:1748-1753.
15. Lichtstein D, Gati I, Samuelov S, et al. Identification of digitalis-like
compounds in human cataractous lenses. Eur J Biochem. 1993;
216:261-268.
16. Fukui HN, Merola LO, Kinoshita JH. A possible cataractogenic
factor in the Nakano mouse lens. Exp Eye Res. 1978;26:477-485.
17. Old SE, Carper DA, Hohman TC. Na,K-ATPase response to osmotic
stress in primary dog lens epithelial cells. Invest Ophthalmol Vis
Sci. 1995;36:88-94.
18. Ikeda U, Hyman R, Smith TW, Medford RM. Aldosterone-mediated
regulation of Na+,K+-ATPase gene expression in adult and neonatal rat cardiocytes./Biol Chem. 1991;266:12058-12066.
19- Farman N, Bonvalet JP, Seckl JR. Aldosterone selectively increases
Na+-K+-ATPase a3-subunit mRNA expression in rat hippocampus.
AmJ Physiol. 1994;266:C423-C428.
20. Garner MH, Horwitz J. Catalytic subunit isoforms of mammalian
lens Na,K-ATPase. Curr Eye Res. 1994;13:65-77.
21. Dean WL, Delamere NA, Borchman D, Moseley AE, Ahuja RP.
Studies on lipids and the activity of Na,K-ATPase in lensfibrecells.
Biochemistry. 1996;3l4:96l-967.
22. Baghieri S, Garner MH. Na,K-ATPase and phospholipid degradation
in bovine and human lenses. Curr Eye Res. 1992; 11:459-467.
23. Garner MH. Na,K-ATPase of the lens epithelium and fiber cell:
formation of catalytic cycle intermediates and Na+:K+ exchange.
Exp Eye Res. 1994;58:705-718.
24. Borchman D, Paterson CA, Delamere NA. Ca2+-ATPase activity in
the human lens. Curr Eye Res. 1989;8:1049 -1054.
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