(Hypertension. 1999;34:1168-1174.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Sodium Pump Inhibition and Regional Expression of Sodium Pump 
-Isoforms in Lens
From the Departments of Medicine (Endocrine-Hypertension Division) and Radiology (N.K.H.), Harvard Medical School, Brigham and Women’s Hospital, Boston, Mass; and the Department of Chemistry (Q.-F.T., S.W.G.) and Biochemistry (S.W.G.), Brigham Young University, Provo, Utah.
Correspondence to Dr Steven W. Graves, Department of Chemistry and Biochemistry, Brigham Young University, BNSN C-212, Provo, UT 84602. E-mail swgraves{at}chemdept@byu.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Both hypertension and cataract formation have been associated with reductions in sodium pump activity, possibly as a result of an endogenous inhibitor. The objective of the present study was to answer 4 closely related questions: (1) Is the lens sodium pump effectively inhibited by a labile, digitalis-like factor we have identified in the peritoneal dialysate from hypertensive patients in end-stage renal failure? (2) How does that inhibition compare to that induced by ouabain? (3) Does sodium pump isoform distribution determine the degree of lens sodium pump inhibition? (This question was precipitated by the unanticipated finding that the labile DLF was more effective in inhibiting lens sodium pump than was anticipated.) (4) Is sodium pump activity altered in lens in response to increased salt intake, a maneuver known to increase endogenous digitalis-like factor? We found that whereas ouabain produced equivalent or significantly less inhibition of lens Na+,K+-ATPase from calf or rabbit, respectively, compared with brain, labile digitalis-like factor preferentially inhibited lens compared with brain. Analysis of whole-lens preparations from rabbit, calf, and normal human lens revealed substantial
2- and 3-isoforms of the sodium pump but
little 1-isoform. Ouabain inhibition of whole-lens
Na+,K+-ATPase from rabbit and calf were
comparable: for rabbit lens,
Ki=5.2x10-7 mol/L; for calf
lens, Ki=1.0x10-6 mol/L.
Limited quantities of labile digitalis-like factor prohibited similar
determinations; however, its concentration-activity profile
paralleled that of ouabain. Na+,K+-ATPase
activity, measured in the 3 major anatomic regions of lens and
normalized to nucleus, was greatest in epithelium (56.9±17.9)
compared with cortex (5.8±1.4) and nucleus (1.0±0.0;
P=0.01). Immunohistochemistry of rabbit lens found
abundant 2- and 3-isoforms in epithelium and limited 3 but
undetectable 1 in cortex and nucleus. Finally, rats randomized to a
high Na diet showed significantly reduced lens
Na+,K+-ATPase activity compared with those on a
low Na diet, consistent with the effects of a sodium pump
inhibitor. In conclusion, the present study suggests
that digitalis-like factor may provide a link between hypertension and
cataract formation.
Key Words: lens • sodium pump • sodium • potassium • ATPase • isoforms • cataracts
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
An association exists between hypertension and human cataract formation,1 and this same relationship occurs in some animal models of hypertension.2 Rodriguez-Sargent et al2 recently reported that development of cataracts in Dahl S rats in response to a high salt diet can be predicted by a reduction in ouabain-sensitive Na+,K+-ATPase activity several weeks before cataract formation. Likewise, a reduction in lens Na+,K+-ATPase (sodium pump) activity occurs in several other animal models of cataract2 3 4 5 and in human cataract formation.3 6 7 8 A similar reduction in sodium pump activity is also a common feature of hypertension and is thought to be mediated in part by a circulating sodium pump inhibitor9 called either digitalis-like factor (DLF) or ouabain-like factor.
These observations led us to address a series of closely related
questions. The first was whether the labile DLF isolated from the
peritoneal dialysate of volume-expanded, hypertensive renal-failure
patients, treated with that modality of dialysis,10
inhibited the sodium pump in lens and how this response compared with
the inhibition produced by ouabain in this and a reference tissue. When
labile DLF was applied to lens, we found it to have an unanticipated
preferential effect on lens compared with brain, a difference not found
with ouabain. This led us to question whether this differential
inhibition could be accounted for by clear differences in sodium pump
-isoform distribution in these tissues. To date, 3 unique isoenzymes
of the catalytically active -subunits of the sodium pump have been
conclusively identified, each coded by a different
gene.11 12 13 A substantial amount of variation in
sensitivity of the sodium pump in different tissues to cardiac
glycosides such as ouabain can be accounted for on the basis of the
isoforms present in that particular tissue. For example, it is now
well recognized that the 1-isoform in rat kidney is highly
"resistant" to ouabain, whereas other sodium pump isoforms
present in rat brain, which has relatively equal amounts of all 3
isoforms, are substantially more sensitive to
ouabain.11
Sodium pump -isoform in lens has previously been studied only in the
rat, and those studies were limited to the
epithelium.14 15 We recognized that differences in sodium
pump sensitivity to labile DLF might be accounted for by isoform
distribution within regions of the lens and might be coupled with in
vivo sensitivity to ouabain and labile DLF. Consequently, we undertook
a systematic assessment of the sodium pump -isoform presence and
distribution in whole lens and within the major structural regions of
lens. We performed immunohistochemistry to determine unambiguously the
location of the various sodium pump isoforms in rabbit lens. Because of
potential species differences, we performed several of our studies in
lens obtained from rabbit, calf, and, where possible, normal humans.
Finally, we also raised the question of whether increased sodium
intake, a maneuver known to increase levels of the
endogenous sodium pump
inhibitor,10 could lead to changes in lens
Na+,K+-ATPase activity in
vivo. The results are reported in the present article.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Lens Preparation
Bovine and rabbit lenses were frozen within one half hour of euthanatization and stored at -70°C (for prolonged periods) or -20°C (for short periods) until use. Rabbits were housed and used locally in compliance with the guidelines of the Committee on Animals of the Harvard Medical School. Bovine lenses were obtained from a local abattoir. Normal human lens was obtained from the New England Eye and Tissue Bank (Boston, Mass) as postmortem research materials representing multiple individuals. Whole-lens or individually dissected anatomic regions of lens were processed into microsomal Na+,K+-ATPase preparations. Animal lenses were removed atraumatically from the enucleated globe and homogenized with several strokes of a glass-Teflon homogenizer in buffer containing (in mmol/L): sucrose 250, histidine 30, and EDTA 1; pH 7.2). The homogenate was pelleted by centrifugation at 15 000g for 30 minutes and then resuspended in the same buffer (0.7 mL for 2 rabbit lenses, 0.8 mL for 2 bovine lenses, and 0.8 mL for 2 human lenses). Human lens was provided as a dissected, intact tissue that was then homogenized, and microsomal preparations were isolated in an equivalent manner.
Production of Labile DLF
Patients with chronic renal failure treated with chronic
ambulatory peritoneal dialysis were enrolled in a Human Subjects
Committee–approved clinical research protocol in which they underwent
gradual sustained volume expansion as described
elsewhere.10 Participation occurred after subjects had
given informed signed consent. Peritoneal dialysate (
2 L) taken
during the period of volume expansion for each subject was
substantially purified by a previously published method that involved
ultrafiltration through a 1000-Da exclusion membrane and 3
high-performance liquid chromatographic
steps.10 Similar volumes of dialysate purified by this
protocol have only 1 active component and have yields in the 0.1- to
0.5-ng range.16 The factor that correlated with volume,
blood pressure, and plasma DLF is the one used in these experiments;
however, this factor is chemically labile and when purified has a
half-life of 10 hours.17 Because of this lability,
specimens could not be pooled; 1 preparation of labile DLF was divided
and used the day it was processed against the tissues studied.
Quantities were insufficient to both measure concentration and also
study tissue inhibition; however, previously, the labile DLF was shown
to be 50 times more potent than ouabain against rabbit kidney (a
non–ouabain resistant source for 1) and 1000 times more
potent than ouabain against rabbit vascular smooth muscle
(predominantly 2).18 This factor has been extensively
characterized in previous publications that show that it acts on the
Na+,K+-ATPase in a
manner analogous to known cardiac glycosides.18
Measurement of Na+,K+-ATPase
Activity
The activity of
Na+,K+-ATPase in animal
lens cell membrane preparations was determined by measuring hydrolysis
of ATP.19 Lens membrane preparation (20 µL) was
incubated for 3 hours in a buffer containing (in mmol/L) Na 100, K
5, Mg 3, EGTA 1, and Tris 80; pH 7.5, 37°C in the absence or presence
of ouabain in graded concentrations. The reaction was started by adding
10 µL of 40 mmol/L [
-32P]ATP
(Amersham) and was ended after 90 minutes by addition of 40 g/L
charcoal in 0.1 mmol/L HCl solution.
Na+,K+-ATPase activity was
defined as that portion of ATPase activity inhibited by 10 mmol/L
ouabain. For comparison, microsomal
Na+,K+-ATPase from rabbit
brain was similarly prepared and ouabain and labile DLF were also
applied to it.
Comparison of the Effects of Labile DLF and Ouabain on Lens
and Brain
Ouabain (10-6 mol/L) was applied to
microsomal preparations of lens
Na+,K+-ATPase from both
calf and rabbit and also rat fetal brain
Na+,K+-ATPase, and
inhibition was measured. A single pool of labile DLF was
simultaneously applied to the same preparation of
Na+,K+-ATPase
microsomes.
Western Blot Analysis
Equal amounts of individual lens membrane preparations from all
3 species (30 µL) were simultaneously run on 7% SDS-PAGE
and transferred to nitrocellulose membrane. For immunodetection of the
isoforms of the sodium pump, the monoclonal antibodies McK1 (specific
for 1-isoform; a gift from Dr Kathleen J. Sweadner, Massachusetts
General Hospital, Harvard Medical School, Boston), McB2 (specific for
2-isoform; also a gift from Dr Sweadner), and MA3-915 (specific for
3-isoform; Affinity BioReagents Inc) were used to bind the
individual isoforms. A second antibody coupled to an enzyme that
converts inactive substrate into chemiluminescent product allowed
for detection (enzyme-linked chemiluminescence; Amersham) and was used
to register the -isoform:first-antibody complex. This procedure was
performed according to manufacturer’s protocol. Each gel was probed
only once for a specific -isoform (ie, we did not reprobe gels), but
the same lens preparations were used for each set of 3 isoforms.
Measurement of -Isoform Distribution and Activity in Regions of
Rabbit Lens
To remove rabbit lens with the capsule intact, the posterior of
the rabbit eyeball was dissected open and the suspensory ligaments of
the lens were cut with fine scissors. The intact lens was then
carefully removed and placed in sterile normal saline solution at room
temperature. Individual lenses were divided into 3 segments as follows:
(1) the superficial (thin) layer, peeled off gently with fine forceps,
which contains the capsule and epithelium (and also a few fiber cells,
because the lens epithelium lies beneath and is tightly attached to the
anterior and equatorial capsule); (2) the median layer, which
represents the soft portion of lens adjoining the superficial
layer containing the cortex and some nucleus; and (3) the center layer,
which represents the residual hard, sticky core and comprises
most of the nucleus of the lens. Each lens layer was individually
homogenized and centrifuged as described above. The
expression of isoforms in these fractions of rabbit lens was evaluated
by Western immunoblot analysis. Equivalent regional
preparations of rabbit lens were also prepared, and
Na+,K+-ATPase activity was
measured for each of the 3 layers of lens tissue as described
above.
Immunohistochemical Detection of the Individual -Subunit of the
Sodium Pump
Immunohistochemistry was performed following a published
method,20 with some modifications. Briefly,
cryostat-produced thin (7-µm) sections of rabbit lens were mounted on
glass slides and fixed with 4% formaldehyde for 5 minutes. The
sections were pretreated with protein block serum-free solution (Dako
Corp) for 30 minutes at room temperature and then incubated with
primary monoclonal -isoform antibody McK1, McB2, or MA3-915
overnight in the same blocking solution in a humidified chamber at
4°C. Next, sections were incubated for 1 hour with a
peroxidase-coupled anti-mouse IgG (Amersham) after these sections were
washed with PBS containing (in mmol/L) NaCl 145 and
Na2HPO4/NaH2PO4
10; pH 7.4). Finally, peroxidase staining was developed for 20 minutes
by use of AEC reagent (Dako AEC substrate system, Dako Corp). The
slides were subsequently mounted with coverslips, and the specimens
were examined by light microscopy. Photomicrographs were taken
(microscope, Carl Zeiss). Frozen sections were used the same day as
prepared or on the next day, after storage at -70°C. Longer storage
periods were found to reduce the staining.
Effects of Dietary Sodium Intake on Rat Lens
Na+,K+-ATPase Activity
Adult Sprague-Dawley rats were randomized to either a high
(1.6% NaCl) or low (0.04% NaCl) salt diet for 5 to 7 days. The
animals were euthanatized. The lens was then rapidly excised and
homogenized in cold homogenization
buffer (see above) using 3 strokes of a glass-Teflon
homogenizer and centrifuged at
15 000g for 30 minutes at 4°C. The protein pellet was
resuspended in 140 µL of assay buffer and a 20 µL aliquot was
placed in each assay tube. After a 30-minute equilibration at 37°C,
the reaction was initiated by adding 10 µL of 40-mmol/L
32P-ATP, allowed to run for 60 minutes, and ended
as described above. Ouabain 10-3 mol/L was added
to other tubes to determine ouabain-sensitive
Na+,K+-ATPase activity,
expressed in micromoles of ATP hydrolyzed per hour per lens.
Statistics
Data are expressed as mean±SE. Comparisons of responses of 2
matched aliquots of inhibitor were performed by paired,
2-tailed Student’s t test. The effects of 2 different
sodium diets were analyzed statistically by unpaired, 2-tailed
Student’s t test. Comparisons among the 3 regions of lens
were performed by ANOVA with post hoc Newman-Keul’s analysis.
A value of P<0.05 was considered statistically
significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Effects of Ouabain and Labile DLF on Preparations of Lens and Brain
Ouabain at 1x10-6 mol/L was presented to microsomal preparations of calf and rabbit lens and also to rat fetal brain Na+,K+-ATPase. Against calf lens, ouabain produced somewhat greater but statistically insignificant inhibition than against brain (lens, n=6, 60.8±12.9% versus brain, n=6, 46.6±7.0% inhibition; P=0.42; Figure 1A). Against rabbit lens, ouabain caused significantly less inhibition than against brain (lens, n=7, 42.4±2.4% versus brain, n=6, 55.8±2.6% inhibition; P=0.01; Figure 1B). In contrast, when equal aliquots of individual preparations of the labile DLF were applied to these same microsomal Na+,K+-ATPase preparations, the labile DLF produced significantly greater inhibition of calf lens than of brain Na+,K+-ATPase activity (lens, n=6, 82.2±9.5% versus brain, n=6, 37.5±4.3% inhibition; P=0.01). Also, in contrast to ouabain, when equal aliquots of individual preparations of labile DLF were applied to rabbit lens Na+,K+-ATPase, there was also greater, but not statistically significant, inhibition compared with brain Na+,K+-ATPase activity (lens, n=7, 55.4±7.8% versus brain, n=7, 42.8±6.5% inhibition; P=0.11). However, for both calf and rabbit, the differences between lens and brain were greater for DLF than for ouabain whether against calf (DLF, 44.6±11.0% versus ouabain, 12.9±14.7%; P=0.047) or rabbit lens (DLF, 11.2±6.4% versus ouabain, -11.2±3.0; P=0.025). These results might be explained by tissue-specific distribution of sodium pump
-isoforms.
|
P<0.05) for both
calf and rabbit lens.
Na+,K+-ATPase -Subunit Expression
in Lens
The distribution of
Na+,K+-ATPase -isoforms
was examined by immunoblot analysis using
monoclonal antibodies specific for the 1-, 2-, or 3-isoform of
the sodium pump after PAGE protein separation.
Representative Western blots are shown in Figure 2. Lens from each of the 3 species
studied showed all 3 -isoforms of the sodium pump. The intensity of
the blot is dependent on both the density of sodium pump units and on
the affinity of the antibody for each isoform in the lens of each of
these 3 species. Nevertheless, abundant 3- and 2-signal was seen
but only a faint immunoreactive band was seen for 1. This pattern
was similar in rabbit, calf, and human lens.
|
Ouabain-Induced Inhibition of
Na+,K+-ATPase Activity
The concentration-dependent inhibition of
Na+,K+-ATPase by ouabain in
lens preparations from rabbit and calf was examined.
Na+,K+-ATPase activity
decreased with increased ouabain concentration, with half-maximal
inhibition achieved in rabbit (n=4; 5.2x10-7
mol/L) or calf lens (n=4; 1.0x10-6 mol/L) at
similar concentrations (data not shown). Although no significant
difference was seen in the affinity of
Na+,K+-ATPase for ouabain
between calf and rabbit lens, the absolute level of enzyme activity in
rabbit lens was significantly higher than in calf lens (0.50±0.07
versus 0.20±0.02 µmol inorganic phosphate per lens per
hour; P<0.02). Although the concentration of the labile DLF
in these experiments is unknown, in part because of its chemical
instability, its dilution-response profile paralleled that of
ouabain closely (data not shown). Previous studies have determined
labile DLF to be 30 to 50 times more active than ouabain against
rabbit and dog 1-isoform and 1000 times more active against the
2-isoform from rabbit than is ouabain.21
Regional Differences in Na+,K+-ATPase
Activity in Rabbit Lens
When Na+,K+-ATPase
activity was determined by region in the rabbit lens, marked
differences were found. Three regions were examined: the superficial
layer containing primarily lens epithelium, a median layer containing
cortex and supranucleus, and a central layer containing the nucleus.
Na+,K+-ATPase activity
measured in each region (expressed per mg tissue) was normalized to the
nuclear Na+,K+-ATPase
activity measured in the same lens. Sodium pump activity was
significantly higher in the superficial layer than in the other layers
(56.9±17.0 for the superficial layer, 5.8±1.4 for the medial
layer, and 1.0±0.0 for the central layer; P=0.01). The
median layer also had somewhat greater activity than the core
(P=0.07; Figure 3).
|
Distribution of -Isoforms in Rabbit Lens
The -isoforms were assessed in rabbit lens by use of the same 3
regions of lens. Of these, only 1 region had detectable expression of
all 3 isoforms, and this area was the surface layer or epithelium. As
shown in Figure 4, the 2-isoform was
predominantly, if not exclusively, found in lens epithelium. Likewise,
the 1-isoform was also detectable only in epithelium, although its
presence was faint. In contrast to 1 and 2, the 3-isoform was
present in all 3 layers of the rabbit lens but with a reduced
density in the cortex and with further reductions in the nucleus. The
pattern observed was easily reproducible. To ensure that the
2-isoform found in the superficial layer was not from ciliary
contamination, a preparation of the ciliary from the same lens was
included on the same gel and blotted. This showed a much weaker signal
for 2 than that seen in the superficial layer and different
nonspecific bands in the gel.
|
Immunohistochemistry of the -Subunits Within Rabbit
Lens
Precise localization of the -isoforms throughout the rabbit
lens was accomplished by immunohistochemistry (Figure 5). A distinct pattern of intense
staining was seen along the lens surface when thin sections were probed
for 2 and 3. However, 1 was not detected. Staining for 2
was confined to the lens epithelium, consistent with data
obtained by Western blot analysis. Although high densities of
the 3 isoform were found in the epithelial layer, it was also
present in the cortex and nucleus in a diminished, diffuse, uneven
distribution (data not shown). Lens incubated with secondary antibody
alone revealed no staining (Figure 5).
|
Effect of Sodium Diet on Na+,K+-ATPase
Activity in Lens
Microsomal preparations of whole lens were rapidly prepared from
rats randomized to 5 to 7 days of a high salt (n=4) or low salt (n=4)
diet. Those that received the high salt diet showed reductions in both
total and ouabain-sensitive
Na+,K+-ATPase activity
compared with lens preparations from animals that received a low salt
diet (total ATPase activity, high salt 0.97±0.16 versus low salt
1.50± 0.11 µmol/h per lens; P=0.03;
Na+,K+-ATPase activity,
high salt 0.53±0.10 versus low salt 0.79±0.04 µmol/h per lens;
P<0.05; Figure 6). No
difference occurred in the degree of ouabain sensitivity of the lens
preparations obtained from rats on the high and low salt diets.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Studies have linked hypertension and cataract formation and both vascular smooth muscle from hypertensive animals and cataractous lens have been characterized by decreased Na+,K+-ATPase activity and increased cell Na content, which may be pathogenic.1 2 3 4 5 6 7 8 The mechanisms for this reduction in Na+,K+-ATPase activity are not completely understood but could result from an endogenous inhibitor or reductions in functional sodium pump units. We have recently isolated a labile, endogenous Na+,K+-ATPase inhibitor from the peritoneal dialysate of hypertensive renal failure patients.10 This factor inhibits the sodium pump in a manner comparable to all known cardiac glycosides22 and causes vascular smooth muscle contraction but is active at much lower concentrations.21 Because such an inhibitor might link and mediate hypertension and cataract formation, it was important to establish that this labile DLF was active against lens Na+,K+-ATPase. As anticipated, labile DLF was an effective inhibitor of the lens sodium pump in calf and rabbit. Moreover, this inhibition was concentration dependent and had a concentration-response profile entirely comparable in pattern to that found with ouabain. Because of the intrinsic chemical instability of the labile DLF, we were unable to accumulate enough material to do the experiments described here and also determine concentrations. Previous studies have demonstrated this factor to be
30 to 50 times more potent than ouabain against rabbit
or dog kidney Na+,K+-ATPase
(1) and 1000 times more potent than ouabain against rabbit
vascular smooth muscle
Na+,K+-ATPase
(predominantly 2); these studies have suggested that this is caused
by DLF having a preferential effect on the 2 isoform.18
This labile DLF also shows no resistance to rat 1, as is found with
ouabain. Comparisons were made of ouabain-induced, sodium pump inhibition in lens versus in rat fetal brain. We used this latter tissue as a reference because it is well characterized in terms of its sodium pump isoform distribution.11 12 The brain sodium pump may also have a role in hypertension, in that it appears to have sodium-regulated increases of a "ouabain-like" factor that may contribute to hypertension in Dahl S rats.23 We found some species differences in the response of ouabain. In calf, ouabain produced a small but insignificantly greater inhibition of lens over brain, but in rabbit, ouabain actually produced significantly less inhibition of lens than brain. The labile DLF against lens from either species showed greater inhibition of lens versus brain. This was statistically significant for calf and nearly significant for rabbit. Interestingly, for either species, DLF produced significantly greater inhibition of lens than did ouabain. Given that the concentration of the labile DLF used against each tissue was identical for 1 set of experiments and that DLF produced less inhibition of brain than ouabain, these results suggest that the lens is more sensitive to labile DLF than to ouabain.
We anticipated that these findings could be explained by differences in
the sodium pump -isoform distribution among lens and brain in the
species studied. We used lens from several species in these studies
primarily because of the difficulty in obtaining normal human lens. The
isoform distribution found for human lens was similar in pattern to
that of calf and rabbit (it had abundant 2 and 3). Within the
lens, the density of sodium pumps was greatest on the epithelial
surface, as determined by studies of protein expression, which showed
that 2 and 1 were found exclusively in the epithelial layer,
whereas 3 was found in all 3 layers but was diminished in internal
regions. More precise localization of the -isoforms in rabbit lens
by immunohistologic techniques confirmed these data and again revealed
that the 2-isoform was found only in the epithelial layer. With this
technique, the 1-isoform was not detected. The 3-isoform was
distributed unevenly throughout the lens, with its highest density also
in lens epithelium. These results would predict that the labile DLF
would be a potent inhibitor of human lens
Na+,K+-ATPase activity.
The species differences for either ouabain or the labile DLF were not
obviously related to -isoform distribution (based on Western
analyses) but more likely reflect the greater absolute
Na+,K+-ATPase activity
found in rabbit; ie, a smaller proportion of pumps in rabbit lens will
be inhibited for a given concentration of inhibitor. The
differences in the effect of the labile DLF compared with ouabain on
lens versus brain might reflect differences in isoform distribution
combined with different affinities of isoform for each of the
inhibitors. However, on the basis of distributions we
found, one might have predicted that the labile DLF, which is not
resistant to rat 1, would be more effective than ouabain
against rat fetal brain, which contains 30% 1 (which is ouabain
resistant). The opposite was found. If the lens were
predominantly 2, this might have explained the preferential effect
of labile DLF (which has been previously shown to preferentially
inhibit 2) on lens over brain. However, the lens obviously has
substantial 3. Unfortunately, Western blot analysis is not
quantitative between species; no method currently exists to measure the
concentration of individual -isoforms, and the individual isoforms
have resisted purification for affinity studies. Hence, our data do not
allow us to determine the absolute quantities of the isoforms in rat as
opposed to calf or rabbit tissue nor describe differences among species
in -subunit affinity for ouabain and labile DLF nor define the
contribution of isoform distribution to the difference.
Our results are generally consistent with those of
others,3 4 8 which have considered the location of
Na+,K+-ATPase activity (but
not isoforms) in human and rat lens. In human lens, ATPase reaction
products were found exclusively in the epithelium.3 8
Kobatashi et al6 measured the activity of
Na+,K+-ATPase in the 3
regions of normal human lens and found a third of the total activity in
the epithelial monolayer, half in the cortical fibers, and the
remainder in the nucleus. Our findings are also similar to those of
more recent studies that have begun to explore sodium pump isoforms in
lens. A study of rat lens epithelium demonstrated the presence of the 3
isoforms in the epithelium.14 Another group performed both
Northern and Western analyses on rat lens epithelium and found
all 3 -isoforms, with 1 staining most intensely.15
This latter finding differs from our results and may reflect species or
antibody affinity differences.
Our results may have implications for lens transparency given that it
is generally accepted that
Na+,K+-ATPase in the lens
epithelial monolayer provides the active transport of monovalent
cations for the whole lens.6 The pattern of sodium pump
localization within the lens suggests that the isoforms may be
specifically regulated and have somewhat specialized functions. Recent
work in other tissues provides evidence for this.26 In
addition to the effects of sodium pump inhibitors on
activity, the sodium pump can be transcriptionally regulated by a
number of hormones.27 Additionally, other hormones can
acutely affect the activity of sodium pump isoforms by initiating
intracellular events that modify the phosphorylation
state of the enzyme.27 Hence, any factor that reduces
sodium pump activity, especially those that preferentially inhibit the
2- or 3-isoforms, may be particularly effective in promoting lens
opacification, and evidence exists for such a role in cataract
formation.3 6 7 8 Studies of human senescent cataract lens
compared with age-matched normal lens demonstrated significant
reductions of epithelial, cortical, and nuclear
Na+,K+-ATPase
activity.6 Other studies have suggested that reductions in
Na+,K+-ATPase activity in
cataractous lens may result from endogenous
inhibitors based on their increased presence in cataractous
lens, although these inhibitors appear to differ from
labile DLF.28 Inhibition of the sodium pump by ouabain
leads to lens opacification in parallel with reductions in sodium pump
activity.24 25 28 Levels of sodium pump
inhibitors increase in parallel with volume in
hypertension.10 The increased incidence of cataracts in
hypertensive patients raises the possibility that both disorders arise
from a common mechanism. Rodriguez-Sargent et al2 found
that cataract development in Dahl salt-sensitive rats was related to
the hypertensive process. We also found lens
Na+,K+-ATPase activity to
be sensitive to dietary sodium intake. Although a sodium pump
inhibitor could account for such findings, this has yet to
be proven. However, such reductions in lens sodium pump activity would
predispose to pathology.
In conclusion, labile DLF is an effective inhibitor of lens
sodium pump activity. Indeed, this factor appears to have substantially
greater effect on lens compared with brain. Given the common finding of
altered sodium pump activity in cataractous lens and the reductions in
lens Na+,K+-ATPase activity
in response to high sodium intake reported in the present study, it
is possible that changes in isoform distribution, number, or
susceptibility to factors that modulate sodium pump activity, including
labile DLF as an effective lens inhibitor, may lead to lens
opacification. Detailed studies of -isoforms in conjunction with the
labile DLF are now needed in cataractous lens.
|
Acknowledgments |
|---|
Parts of this research were funded by Marion Merrell Dow (now Hoechst Marion Roussell). We thank Dr Kathleen Sweadner, Massachusetts General Hospital, Boston, for her generous gift of antibodies that made this work possible.
Received May 25, 1999; first decision June 21, 1999; accepted July 12, 1999.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Klein BEK, Klein R, Jensen SC, Linton KLP. Hypertension and lens opacities from the Beaver Dam Eye Study. Am J Ophthalmol. 1995;19:640–646.
2.
Rodriguez-Sargent C, Estape ES, Fernandez N, Irizarry
JE, Cangiano JS, Candia OA. Altered lens short-circuit current in adult
cataract-prone Dahl hypertensive rats. Hypertension. 1996;28:440–443.
3. Ahmad SS, Tsou KC, Ahmad SI, Rahman MA, Kirmani TH. Studies on cataractogenesis in humans and rats with alloxan-induced diabetes. Ophthalmic Res. 1985;17:1–11.[Medline] [Order article via Infotrieve]
4.
Unakar NJ, Tsui JY. Sodium-potassium-dependent ATPase
1: cytochemical localization in normal and cataractous rat lenses.
Invest Ophthalmol Vis Sci. 1980;19:630–641.
5. Unakar NJ, Tsui JY, Kuck JF, Kuck KD. Sodium-potassium-dependent-ATPase activity in Emory mouse lens. Curr Eye Res. 1986;5:263–271.[Medline] [Order article via Infotrieve]
6. Kobatashi S, Roy D, Spector A. Sodium/potassium ATPase in normal and cataractous human lenses. Curr Eye Res. 1982;2:327–334.[Medline] [Order article via Infotrieve]
7. Baghieri S, Garner MH. Na, K-ATPase and phospholipid degradation in bovine and human lenses. Curr Eye Res. 1992;11:459–467.[Medline] [Order article via Infotrieve]
8. Laursen AB, Klauber A, Jensen OA. Human senile cataract and Na-K ATPase activity in the anterior lens structures. Acta Ophthalmol (Copenh). 1980;58:496–505.[Medline] [Order article via Infotrieve]
9. Haddy FJ. The role of a Na+, K+-ATPase inhibitor in regulating precapillary vessel tone. J Cardiovasc Pharmacol. 1984;6(suppl 2):S439–S456.
10. Glatter KA, Graves SW, Hollenberg NK, Soszynski PA, Tao Q-F, Frem GJ, Williams GH, Lazarus JM. Sustained volume expansion and [Na, K]ATPase inhibition in chronic renal failure. Am J Hypertens.. 1994;7:1016–1025.[Medline] [Order article via Infotrieve]
11. Sweadner KJ. Isoenzymes of the Na+/K+-ATPase. Biochim Biophys Acta. 1989;988:185–220.[Medline] [Order article via Infotrieve]
12. Lingrel JB, Orlowski J, Shull MM, Price EM. Molecular genetics of Na, K-ATPase. Prog Nucleic Acid Res Mol Biol. 1990;38:39–89.
13.
Herrara VLM, Emanuel JR, Ruiz-Opazo N, Levenson R,
Nadal-Ginard B. Three differentially expressed Na, K-ATPase
subunit isoforms: structural and functional implications. J
Cell Biol. 1987;105:1855–1865.
14. Garner MH, Horwitz J. Catalytic subunit isoforms of mammalian lens Na, K-ATPase. Curr Eye Res. 1994;13:65–77.[Medline] [Order article via Infotrieve]
15.
Moseley AE, Dean WL, Delamere NA. Isoforms of Na,
K-ATPase in rats lens epithelium and fiber cells. Invest
Opthalmol Vis Sci. 1996;37:1502–1508.
16. Xie LQ, Markides KE, Lee ML, Hollenberg NK, Williams GH, Graves SW. Bioanalytical applications of multidimensional open tubular column supercritical fluid chromatography. Chromatographia. 1993;35:363–371.
17. Graves SW, Soszynski PA, Tao QF, Williams GH, Hollenberg NK. A labile endogenous Na-pump inhibitor in man. In: Bamberg E, Schoner W, eds. The Sodium Pump: Structure, Mechanism, Hormonal Control, and Its Role in Disease. Darmstadt, FRG: Steinkopff Verlag; 1994: 771–774.
18.
Tao Q-F, Hollenberg NK, Price DA, Graves SW. Sodium
pump isoform specificity for the digitalis-like factor isolated from
human peritoneal dialysate. Hypertension. 1997;29:815–821.
19. Tao Q-F, Soszynski PA, Hollenberg NK, Graves SW. A sensitive [Na, K]ATPase assay specific for inhibitors acting through the digitalis-binding site. J Cardiovasc Pharmacol. 1995;25:859–863.[Medline] [Order article via Infotrieve]
20. Kifor O, Moore FD Jr, Wang P, Goldstein M, Vassilev P, Kifor I, Hebert SC, Brown EM. Reduced immunostaining for the extracellular Ca2+-sensing receptor in primary and uremic secondary hyperparathyroidism. J Clin Endocrinol Metab. 1996;81:1598–1606.[Abstract]
21. Graves SW, Tao Q-F, Markides KE, Williams GH, Hollenberg NK. A labile sodium pump inhibitor from the peritoneal dialysate of hypertensive renal failure patients: Estimates of potency. Clin Exp Hypertens. 1998;20:611–616.
22. Tao QF, Soszynski PA, Hollenberg NK, Graves SW. Specificity of the volume-sensitive sodium pump inhibitor isolated from human peritoneal dialysate in chronic renal failure. Kidney Int. 1996;49:420–429.[Medline] [Order article via Infotrieve]
23.
Leenen FHH, Hansen E, Yu H. Dietary sodium and central
vs peripheral ouabain-like activity in Dahl salt-sensitive
vs salt-resistant rats. Am J Physiol. 1994;267:H1916–H1920.
24.
Kinoshita JH. Mechanisms initiating cataract formation.
Invest Ophthalmol. 1974;13:713–724.
25. Mayman CI, Miller D, Tilerina ML. In vitro production of steroid cataract in bovine lens. Acta Ophthalmol (Copenh). 1979;57:1107–1115.[Medline] [Order article via Infotrieve]
26.
Juhaszova M, Blaustein MP. Na+
pump low and high ouabain affinity alpha subunit isoforms are
differently distributed in cells. Proc Natl Acad Sci
U S A. 1997;94:1800–1805.
27.
Ewart HS, Klip A. Hormonal regulation of the
Na+-K+-ATPase: mechanisms
underlying rapid and sustained changes in pump activity. Am
J Physiol. 1995;269:C295–C311.
28. Lichtstein D, Gati I, Samuelov S, Berson D, Rozenman Y, Landau L, Deutsch J. Identification of digitalis-like compounds in human cataractous lenses. Eur J Biochem. 1993;216:261–268.[Medline] [Order article via Infotrieve]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||