The Effects of Digitalis-like Compounds on Rat Lenses
David Lichtstein,1 Talia Levy,1 Joseph Deutsch,2 Michael Steinitz,5 J. Samuel Zigler,Jr,4 and
Paul Russell4
PURPOSE. Fundamental
to the maintenance of ionic concentration gradients and transparency of the
lens is the activity of Na+,K+-adenosine triphosphatase (ATPase) in the epithelial layer. Recent
studies have identified endogenous digitalis-like compounds (DLCs) and 19-norbufalin and its
peptide derivatives in human cataractous lenses. These compounds inhibit the activity of Na^K"*"ATPase and have been suggested to be involved in cataract formation. The present experiments
were designed to test this hypothesis by determining the ability of digitalis and DLCs to induce
changes in protein composition and leakage from rat lenses in organ culture.
were determined in rat lenses using three independent assays: interaction with
ouabain antibodies, interaction with bufalin antibodies, and inhibition of [3H]-ouabain binding to
red blood cells. Rat lenses were incubated in modified TC-199 medium in 5% CO2 atmosphere at
37°C for the time of the experiment. The onset of cataractogenesis was assessed by measuring
protein leakage from lenses and by crystallin composition in the lens and media.
METHODS. DLCS
were present in rat lens with concentrations 7 to 30 times higher in the capsularepithelial layer than in the lens fibers regions. Ouabain, bufalin, digoxin, and DLC induced dose- and
time-dependent leakage of protein from rat lenses. Lenses incubated with these compounds
showed alterations in crystallin content consistent with changes that initiate opacity. All the
compounds caused a multilayering of epithelial cells in the region surrounding the mitotic area and,
at the same time, cell death in the central anterior region.
RESULTS. DLCS
Digitalis and endogenous DLCs are cataractogenic factors. These results, together
with the demonstration of DLCs in the normal lens and their increased levels in human cataractous
lenses, strongly suggest their involvement in the molecular mechanisms responsible for cataract
formation. (Invest Ophthalmol Vis Sci. 1999;40:407-4l3)
CONCLUSIONS.
odium and potassium-adenosine triphosphatase (Na+,K+ATPase, E.C. 3-6.1.3) activity is widely regarded as the
cellular sodium-potassium pump. The enzyme hydrolyzes adenosine triphosphate and uses the free energy to drive
the transport of potassium into the cell and sodium out of the
cell, against their electrochemical gradients.1 Cardiotonic steroids such as ouabain or digoxin bind specifically to the a
subunit of Na+,K+-ATPase, and this interaction results in the
inhibition of enzyme activity and Na+ and K+ transport.2
Because most cardiotonic steroids are obtained from plants and
have been used therapeutically, this interaction was considered to be important pharmacologically, but not significant in
normal physiologic regulation of the Na+,K+-ATPase. It has
been suggested, however, that the binding site for cardiotonic
steroids is actually a receptor for unidentified endogenous
S
From the ' Department of Physiology, 2 Department of Pharmaceutical Chemistry, 3 Department of Pathology, The Hebrew University,
Hadassah Medical School, Jerusalem, Israel; and the ^Laboratory of
Mechanisms of Ocular Diseases, National Eye Institute, Bethesda, Maryland.
Supported by grants from the Israel-U.S.A. Binational Science
Foundation, Jerusalem, Israel, and the United States National Research
Council, Washington, D.C.
Submitted for publication March 24, 1998; revised July 1, 1998;
accepted August 14, 1998.
Proprietary interest category: P.
Reprint requests: David Lichtstein, Department of Physiology, The
Hebrew University, Hadassah Medical School, PO Box 12272, Jerusalem 91120, Israel.
digitalis-like compounds (DLCs).3"5 Recently, several laboratories have identified steroidal DLCs in animal tissues. Ouabain
has been identified in human plasma6 and bovine adrenal
glands,7 digoxin has been shown to be present in human
urine,8 a ouabain isomer has been identified in bovine hypothalamus,9 and 19-norbufalin and its peptide derivative were
identified in the nuclear region of human cataractous lenses.10
In addition, marinobufagenin-like immunoreactivity1' and
proscillaridin A-like immunoreactivity7 have been shown in
human plasma.
In the lens, evidence for an ouabain-sensitive Na^K"*"ATPase has been shown through biochemical12 and histochemical13 experiments. It was shown that the lens Na+,K+-ATPase
maintains intracellular Na+ and K+ concentrations similar to
those in other tissues.14 Characterization of the lens Na+,K+ATPase is of considerable interest because of the important
role played by this enzyme in the inward transport of K+ and
outward transport of Na+ in epithelial and fiber cells.15 Although Na+,K+-ATPase activity is concentrated in the cortical
and equatorial layers of the lens, reduced activity was also
reported in the fiber cells of the nucleus.16"17
Several studies raised the possibility that an Na+,K+-ATPase inhibitor is involved in cataract formation. In I960 a strain
of mice in which lens opacity develops soon after birth was
described by Nakano et al.18 Further investigation of the mechanism of the development of this hereditary cataract suggested
that an apparent deficiency of Na+,K+-ATPase in the lens may
be involved.19 The continuation of this research led to the
Investigative Ophthalmology & Visual Science, February 1999, Vol. 40, No. 2
Copyright © Association for Research in Vision and Ophthalmology
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407
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Lichtstein et al.
isolation of a cataractogenic factor from mice lenses that inhibits Na+,K"t'-ATPase activity.20"21
The recent demonstration that human cataractous lenses
contain relatively high levels of DLC, 19-norbufalin, and 19norbufalin-Thr-Gly-AL,10 together with the established role of
Na+,K+-ATPase in lens function, prompted us to evaluate the
possible effects of digitalis and the newly synthesized DLCs on
lens integrity and in cataract formation. Although the effect of
ouabain on protein leakage from the lens has been studied
before,22 we have extended this study by comparing its effect
on lens integrity with the effect of the endogenous DLC and
another cardenolide (digoxin) and bufadienolide (bufalin).
METHODS
Extraction and Determination of DLCs
A mixed population (male and female) of Sprague-Dawley rats
was used in this study (Taconic, Germantown, NY). We strictly
adhered to the guidelines set forth in the National Institutes of
Health Guide for the Care and Use of Laboratory Animals and
the ARVO Statement for the Use of Animals in Ophthalmic and
Vision Research. Rats (75-100 g) were killed, and lenses were
immediately dissected. The capsule and epithelium were separated from the remaining lens. Using a dissecting microscope,
rat lenses were dissected from the globe. The posterior capsule
was grasped with sharp forceps and split. The capsule with the
epithelium attached was stripped from the remaining lens.
Although the tips of some fiber cells probably adhered to the
capsule and epithelium, visible adhering fiber cells were not
noted. DLCs were extracted by homogenizing the tissue with
three strokes in a homogenizer (Tekmar, Cincinnati, OH) in the
presence of 20 volumes of ethanol. After 1 hour at 4°C the
samples were centrifuged at 15,000,g and the supernatant separated and evaporated to dryness. The samples were dissolved
in 200 /LLI water and 20-JLLI aliquots were used for the determination assays. The DLCs were determined by three independent quantitative bioassays: Inhibition of [3H]-ouabain binding
to red blood cells (RBCs); competitive inhibition enzymelinked immunosorbent assay (ELISA) based on anti-ouabain
antibodies, and competitive inhibition ELISA using anti-bufalin
antibodies.
Inhibition of [3H]-Ouabain Binding to RBCs
Inhibition of [3H]-ouabain binding to RBCs was performed as
described previously23: 250 /xl RBC suspension was transferred
to incubation tubes containing a 200-/xl sample (or ouabain
standard). The samples were incubated for 2 hours at 37°C
with shaking. After the preincubation, 50 /xl [3H]-ouabain (19-5
Ci/mmol, 40,000 disintegrations per minute, 80 nM; New England Nuclear, Dreieich, Germany) were added, and the samples were incubated for an additional 2 hours at 37°C. The
reactions were terminated by the addition of 1 ml 0.9% NaCl
and centrifugation at l680g for 10 minutes. The RBC pellet was
washed twice in 0.9% NaCl, and the supernatant was decanted.
The pellets were suspended in 250 /xl 10% trichloroacetic acid
and mixed vigorously. After 10 minutes at 4°C, aliquots of 200
/xl were assayed for radioactivity. In each experiment, dosedependent inhibition of ouabain displacement of [3H]-ouabain,
using standards of known ouabain concentrations, was performed to calibrate the assay.
IOVS, February 1999, Vol. 40, No. 2
Conjugation Procedures
Ouabain-bovine serum albumin (BSA) conjugate, subsequently used to immunize rabbits, was prepared as described
by Masugi et al.,24 and the ouabain-ovalbumin used in die
ELISA was prepared as described by Harris et al.25 The conjugation of bufalin to BSA was performed by generating bufanolide hemisuccinate and then reacting it with BSA, as described
previously.23
Preparation of Rabbit Anti-Ouabain Serum
A total of 500 mg ouabain-BSA in complete Freund's adjuvant
was injected intramuscularly and subcutaneously into 3- to
4-month-old rabbits. The animals were rechallenged as described seven times at 6-week intervals, with the same amount
of antigen emulsified in incomplete Freund's adjuvant. The
anti-ovalbumin titers of the hyperimmune rabbit sera were low
compared with the anti-ouabain titers and were undetectable
at the dilution used.
Ouabain ELISA
A sensitive, competitive-inhibition ELISA was designed for the
quantification of soluble ouabain. In this assay, samples were
tested for their ability to inhibit the specific binding of rabbit
antibodies to solid-phase bound ouabain. Each well in an ELISA
plate (microELISA; Maxisorp SurFace; Nage Nunc, Denmark)
was coated overnight at room temperature with 200 /xl conjugated ouabain-ovalbumin (0.2 /xg/ml) in 0.04M NaHCO3 (pH
9.8). The antigen solution was then removed, and the wells
were saturated for 1 hour at room temperature with 250 /xl
phosphate-buffered saline (PBS) containing 50% heat-inactivated fetal calf serum.
A 125-ju-l volume of anti-ouabain rabbit serum (diluted
1:12,000 in PBS) was first mixed with one equal volume of
sample containing an unknown concentration of ouabain-like
material and preincubated overnight at room temperature.
Two hundred microliters of this mixture was then added to
each of the ouabain-ovalbumin- coated ELISA wells for 1 hour,
and wells were washed three times with 200 /xl PBS-gelatin
(PBS [pH 7.4] containing 0.15% gelatin and 0.05% Tween 20).
A 200-/xl volume of alkaline phosphatase- conjugated goat antirabbit IgG (diluted 1:20,000 in PBS-gelatin) was added, and the
plates were incubated for 1 hour at room temperature. The
wells were then rewashed, 200 /xl/j-nitrophenyl phosphate (1
mg/ml in 1 M diethanolamine buffer [pH 98] containing 0.5
mM MgCl2 and 0.05% NaN3) were added to each well, and the
plates were incubated for 2 hours. Absorbance at 405 nm was
determined in an ELISA reader (MR 5000; Dynatech, Burlington, MA). The values were obtained and compared with those
for noninhibited binding of the diluted anti-ouabain serum, and
the percentage of inhibition was calculated. Sample concentration was determined according to a standard ouabain-inhibition curve in the range 10~4 M to 10~12 M.
Bufalin ELISA
The assay used to quantitate bufalin was similar to the one
described for ouabain, with only minor modifications. The
rabbit serum used in the bufalin test was a gift from Vincent P.
Butler, Department of Medicine, College of Physicians and
Surgeons, Columbia University, New York, New York. The
antibodies were raised against a bufalin-BSA conjugate, and the
ELISA wells were coated with that antigen. The hyperimmune
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IOVS, February 1999, Vol. 40, No. 2
TABLE
Digitalislike Compounds and Cataract
409
1. Digitalis-like Compounds in Capsular and Fiber Cell Regions of Rat Lens
[3H]-Ouabain-Binding
Inhibitor
Capsular
layer
Decapsulated
lens
Ouabain ELISA
Bufalin EUSA
Picomoles/g
Picomoles/
Sample
Picomoles/g
Picomoles/
Sample
Picomoles/g
Picomoles/
Sample
143.2 ± 4lt
5.6 ± 1.1
192.7 ± 84*
7.6 ± 2.3
74 ± 31*
3.07 ± 1.2
6.0 ± 1.1
6.4 ± 1.9
22.8 ± 16.2
1.69 ± 0.44
9±3
2.41 ± 0.85
ELISA, enzyme-linked immunosorbent assay.
Results are expressed in picomoles per sample (12 lenses) and picomoles per gram wet tissue, as mean ± SEM of 11 determinations. Extraction
procedure, [3H]ouabain binding to red blood cells assay, ouabain ELISA, and bufalin ELISA were conducted as described in the Materials and
Methods section.
Significantly higher level than in decapsulated lens.
* P < 0.05.
t / > < 0.01.
serum was diluted in PBS containing 1% BSA to avoid binding
of the anti-BSA antibodies to the solid-phase bound conjugate.
Effect of Digitalis Compounds on Lens Integrity
For organ culture, rat lenses were dissected from the globe,
and each lens was incubated separately in 2 ml modified
TC-199 medium at 37°C in 5% CO2 atmosphere according to
the method of Zigler and Hess.26 Stock solutions of ouabain,
bufalin, digoxin, and DLC were prepared in ethanol and diluted
into the wells so that the final alcohol concentration did not
exceed 1%. The DLC used in this study was a synthetic 19norbufalin derivative. The synthesis procedure will be published elsewhere (JD and DL, manuscript in preparation). Protein levels in the medium were determined by a modification of
the Bradford method.27 The protein leakage was shown to be
a quantitative and sensitive assay for the determination of lens
integrity.
Electrophoretic and Immunoblotting Analysis
After organ culture, the culture media were taken and trichloroacetic acid was added to make afinalconcentration of 10%.
The sample was centrifuged at l4,000g for 15 minutes, and the
supernatant was decanted. The pellet was solubilized in gel
sample buffer (62.5 mM Tris-HCl [pH 6.8] with 2% sodium
dodecyl sulfate). Protein concentrations were determined by
the bicinchoninic acid (BCA) assay (Pierce Chemical, Rockville, IL). The rat lenses were homogenized in 8 M urea and 2%
Nonidet P-40 (American Bioanalytical, Natick, MA) to obtain a
urea-soluble fraction. Protein concentrations were determined
with the Bradford assay. Proteins (3 ju-g) were subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) on 12.5% gels using a commercial system (PhastGel; Pharmacia LKB Biotechnology, Piscataway, NJ). The gels
were silver stained. Polypeptides were transferred to nitrocellulose membranes according to the manufacturer's protocol
and blocked for 1 hour. Membranes were incubated with
rabbit polyclonal antibodies to a-crystallins, j3-crystallins, and
y-crystallins, and the blots were subsequently incubated with a
chemiluminescence system (CSPD; Tropix, Bedford, MA).
Histology
Rat lenses were fixed for 1 hour in 2.5% glutaraldehyde in
cacodylate buffer (pH 7.2) containing 4% sucrose and 2 mM
CaCl2. The lenses were then transferred to 10% buffered formalin for 2 days and were embedded in paraffin. Sections were
stained with hematoxylin and eosin.
RESULTS
DLCs in Rat Lens
DLCs were present in normal transparent rat lenses (Table 1).
The levels of DLC in the epithelial-capsular layer were 7 to 30
times higher than the levels in the lens fiber regions of the lens
(Table 1). Despite the variability in DLC levels, mainly in the
capsular layer of the rat lens, the increased level compared
with the levels in the lens fiber layers was statistically significant. Although the three assays were measuring different entities of DLCs (i.e., interaction with die ouabain-binding site on
Na+,K+-ATPase, and interactions with anti-ouabain and antibufalin antibodies) the levels obtained by the three assays were
similar. These data support the assertion that the three methods determine a similar chemical entity. Thus, the presence of
DLCs was clearly established in normal nit lens.
Effect of Digitalis on Lens Integrity
Digitalis and DLCs when added to the culture media caused
leakage of protein from the lens. An example of the resultant
effect of ouabain, bufalin, digoxin, and DLCs on rat lens integrity is shown in Figure 1. This effect was dose and time
dependent. In the example depicted, a significant increase in
protein leakage resulting from exposure to a 10 yM concentration of each drug tested was achieved after 29 hours of
incubation. At 100 /LIM all drugs induced protein leakage after
16 hours, and at 100 nM significant leakage was obtained after
approximately 48 hours of incubation (data not shown). In all
experiments bufalin was the most potent inducer of protein
leakage, causing the earliest and the highest release of protein
into the culture medium (Fig. 1).
In the control medium, the only protein band apparent on
silver-stained SDS-PAGE gel was albumin (Fig. 2). The presence
of a small amount of albumin in the culture medium is consistent with that observed in previous work.29 In the medium
from the treated lenses, we confirmed the presence of immunoreactive a-crystallins, j3-crystallins, and y-crystallins (Figs.
2A, 2B, 2C, respectively). In addition to the presence of the
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10VS, February 1999, Vol. 40, No. 2
160
120
80
40
0
0
10
20
30
40
50
60
TIME (hours)
FIGUKE 1. Effect of digitalis and digitalis-like compounds (DLCs) on protein leakage from rat lenses. Rat
lenses were incubated in modified TC-199 media, as described in the Methods section. After the addition
of the drugs, 30 /xl media was removed at the indicated times, and protein levels were determined. The
effect of 10 juM of ouabain ( • ) , bufalin (A), digoxin (•), DLC (D), and control media ( • ) are shown. Each
point represents the mean ± SEM of 6 to 12 lenses. Seven additional experiments were conducted with
similar results.
crystallins migrating in the region of approximately 20 kDa to
28 kDa, several other bands having lower molecular weights
were evident. It was presumed that these bands represented
degradation products of the crystallins, although not all reacted
with the three antisera tested.
Characteristic changes in the crystallin patterns in lenses
treated with DLCs were apparent after 1 day in organ culture
(Fig. 3). When the lens proteins are run on SDS-PAGE, a loss of
the j3Bl-crystallin band (Fig. 3, top arrow) was evident with the
appearance of the /3B1b band that is located just below the
unmodified form. This can be seen particularly well in the lens
treated with ouabain where the two bands appeared, one
directly under the other. With the other treatments, the /3B1
kDa
6843-
band was lost completely, and only a small band remained in
the j3Blb position. Another obvious alteration was the appearance of degraded polypeptides (Fig. 3, lower arrow) in the
treated lenses. Both of these major alterations in the crystallin
patterns have been observed in a number of rodent cataracts
and are indicative of the macroscopic changes that occur in the
lens crystallins with opacification.30
Lenticular integrity was also examined using conventional light microscopy. To our knowledge, this is the first
examination of the effects of digitalis compounds on lens
integrity using this tool. Lenses incubated in the presence of
ouabain, bufalin, and DLC showed obvious changes consistent with cataractogenesis (Figure 4), An intriguing change
A
B
30- r
20- !
m
1
FIGURE 2. One-dimensional gel of proteins in the media from organ-cultured lenses after 3 days detected
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The media samples are Lane 1, control;
lane 2, 10 JLIM ouabain treated; lane 3, 10 p,M bufalin treated; lane 4, 10 JLLM digoxin treated; and lane 5,
10 (xM 19-norbufalin derivative treated. Western blot analyses (right) are shown for the boxed area of the
gel, using antibodies to a-crystallins (A), /3-crystallins (B), and y-crystallins (C).
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Digitalis like Compounds and Cataract
10VS, February 1999, Vol. 40, No. 2
4li
the epithelial cells in the central area were lost (Fig. 5). At
day 2, control lenses had some vacuoles present in the
epithelial cells in this region, whereas the treated lenses
showed areas where no cell nuclei were present. Again, the
degree of damage to the epithelial cells appeared consistent
with the rate of protein leakage from the treated lenses.
DISCUSSION
FIGURE 3. One-dimensional gel of protein from organ-cultured lenses
detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Samples are from lenses treated for 3 clays. Lane J, control; lane 2,
10 fxM ouabain treated; lane 3, 10 /u,M bufalin treated; lane 4, 10 ji,M
digoxin treated; and lane 5, 10 /xM 19-norbufalin derivative treated.
Arrows point out the loss of fJBI-crystallin at 31 kDa in the treated
lenses and the increase in degraded proteins below 20 kDa.
was the multilayering of the epithelial cells in the area
anterior to the equator. This effect was pronounced in the
bufalin-treated lens as early as 1 day but was found with
lenses treated with ouabain and DLC as well at later time
points. By the third day, there was a total loss of the cells in
this area in the bufalin-treated lenses and a partial loss of
cells in the DLC-treated lenses. The changes in the structural
integrity of the lens induced by the drugs are in accord with
protein leakage data (Fig. 1), with bufalin being the most
damaging followed by the 19-norbufalin derivative, and then
by ouabain. In addition to multilayering, swelling of the cells
in the equatorial and bow areas also occurred. At the posterior pole by day 3, the swelling of the fiber cells was
evident in all the treated lenses. Of interest, while the cells
in the area just anterior to the equator underwent mitosis,
CONTROL
Steroidal DLCs, resembling the structure of plant cardenolides
(ouabain and digoxin) and toad bufaclenolides (bufalin) have
been identified as normal constituents of human and animals
plasma, adrenal glands, and brain. 6 "" Although the metabolic
pathway for the biosynthesis of these compounds has not yet
been fully elucidated, it has been shown that they are synthesized by the steroidogenic pathway in the adrenal glands.2331
We showed here for the first time that the DLCs present in the
rat lens were localized mainly in the capsule-epithelium layer
of this organ. This increased concentration of DLC in the
capsular layer is in agreement with a previous study using
bovine lenses. In addition, this was the first demonstration that
the DLCs extracted from lens tissue are structurally related to
and can be quantitatively determined using antibodies to
ouabain and bufalin.
Of note, although DLC levels in rat lens fibers were similar
to the levels in the bovine lens, the level in the capsular layer
was significantly lower than that in the capsular layer of the
bovine lens (143.2 ± 41 picomoles/g versus 611 ± 181 picomoles/g in the rat and bovine lenses, respectively).17 The levels
of DLCs in the lens fibers determined in this study were
significantly lower than DLC levels determined previously in
whole rat lens extract.10 The different methods for DLC determination used in these studies (inhibition of [3H]-ouabain in rat
brain synaptosomes versus in human red blood cells) may be
the reason for this discrepancy. The levels of DLC in the rat
lens determined in this study were in the same order of mag-
OUABAIN
BUFALIN
19-NORBUFALIN
DERIVATIVE
ONE DAY
TWO DAYS
THREE DAYS
POSTERIOR POLE
THREE DAYS
FIGURE 4. Histologic appearance of lenses incubated with 19-norbufalin derivative and other digitalis compounds. The lenses were treated with
1 mM ouabain, 1 mM bufalin, and 100 ixM 19-norbufalin derivative. The leftmost column shows control lenses in the region of the lens bow at
1,2, and 3 days of incubation. The last row in the figure is the region at the posterior pole on day 3.
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Control
IOVS, February 1999, Vol. 40, No. 2
Ouabain
Bufalin
19-Norbufalin
Derivative
5. Histologic appearance of the central anterior section of the lens treated for 2 days. Left to right.
Control lenses, 1 mM ouabain treated, 1 mM bufalin treated, and 100 fxM 19-norbufalin derivative.
FIGURE
nitude as the levels in the rabbit, cat, and bovine lenses and
were significantly lower than the levels in the nuclear region of
cataractous human lenses.M) Furthermore, because DLC levels
in other tissues such as kidney, liver, heart, and brain are in the
range of 2 to 10 picomoles/g6 the increased levels in the lens
capsular-epithelial layer is of special interest and deserves further attention. The possibility that the increased levels resulted
from increased synthesis in the epithelium, or alternatively,
from their specific accumulation in this tissue is currently
under investigation.
The importance of normal osmotic balance for maintaining transparency of the lens is widely recognized. During the
formation of many types of cataract, the lens uptake of Na+ is
associated with loss of K+.32 The changes in monovalent cation content and composition may result from decreased
Na^K^-ATPase activity, increased membrane permeability, or
a combination of both. Loss of Na+,K+-ATPase activity has
been reported to occur in the hereditary cataracts in the
Nakano mouse18, in the Emory mouse,29 and during cataract
formation in the galactose-fed rat*3 and the triparanol-fed rat.34
Most human age-related cataracts show a progressive alteration
of electrolyte levels resulting in increased lens sodium and
decreased potassium and a concomitant increase in lens water/ 5 Na^.K^-ATPase in these human cataracts has been studied with contradictory results, with increased36 and reduced
activities37 reported. The results of the present study show that
the inhibition of Na+,K+-ATPase by digitalis compounds initiated cataract formation manifested by crystallin degradation
and protein leakage from the lens and by changes in the
structural integrity of the lens. This result is in accord with the
hypothesis that the endogenous DLCs play a role in the mechanisms responsible for cataract development. The changes in
crystallin composition of the lenses after their treatment with
DLC and digitalis were simitar to those described in other
cataracts such as those in the Nakano mouse and in the HTV-1
protease transgenic mouse,18'29 in which opacification was
induced by other perturbations.
The higher potency of bufalin as an inducer of cataract can
be explained by the high potency of this compound as an
inhibitor of Na+,K+-ATPasc activity.3** In rat brain synaptosomal preparations and microsomal preparations from various
tissues of die toad37 bufalin was 10 times more effective an
inhibitor of the Na+,K+-pump than were ouabain or digoxin.
We showed in this study that the inhibition of the Na+,K+pump not only caused cell swelling and cell death, as shown by
the cells in the bow region and anterior epithelium, but that it
also triggered mitosis in some cells. Although the ability of
these inhibitors to cause certain cells to undergo mitosis has
been documented,39 this is the first demonstration that DLCs
can influence lens cells in this way. Again, the rate at which the
inhibitors initiated cell division matched their ability to inhibit
the pump. The multilayering was apparent in the bufalintreated lenses by the first day, whereas it was just starting to
occur in the ouabain-treated lenses by the third day.
The molar concentration of DLCs in the lens was in the
range of 100 11M to 300 nM in the capsular- epithelial layer and
10 nM to 30 nM in the fiber cells (assuming 60% of water
content). Protein leakage was induced by DLCs at concentrations as low as 1 juiM. Thus, the concentrations of DLC that
produced lens damage in this study are 10 to 100 times higher
than the concentrations found in the capsular or fiber cells
layers, respectively. In the earlier studies with the human
lenses, only the cataractous nucleus could be measured for
DLCs. Currently, we are attempting to collect data about levels
of DLCs in the various regions of donor lenses, with and
without frank opacities. Our hypothesis is that the levels are
higher in the superficial regions of the lens than in other
regions closer to the nucleus. Because age-related cataract
formation is time dependent, we hypothesize that older lenses
that have been subjected to insults such as oxidation and UV
radiation may not be able to adjust to the inhibition of Na"*",K+ATPase caused by the presence of these DLCs. Gradually, with
a change in ion composition in the cells, synthesis of components essential to lens homeostasis will decrease. The progression of lens cell damage then intensifies, and cataractogenesis
results. The manifestation of these cataracts is conjecture at
this point. Although we favor a model suggesting the more
gradual loss of function in the lens resulting in a nuclear
cataract, in some lenses in which a high level of DLCs is
present, a cortical cataract may result. The rat model system is
useful for showing the presence and quantity of DLCs endogenous to the lens and provides a way to demonstrate the
potential that these compounds have to cause opacification.
These data confirm previous observations that the inhibition of
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JOVS, February 1999, Vol. 40, No. 2
Na^.K^-ATPase leads to lens opacification and argues for the
need for further biochemical studies to test the possibility that
endogenous DLCs are involved in the cause of cataract formation.
References
1. SkouJC, NorbyJG, Maunsbach AB, Esmann M. The Na+,K+-pump,
New York: Alan R. Liss; 1988.
2. Hoffman BF, Bigger JT Jr. Digitalis and allied cardiac glycosides, In:
Goodman-Gilman A, Rail TW, Nies AS, Taylor P, eds. The Pharmacological Basis of Therapeutics. 8th ed. New York: Pergamon
Press; 1990:814-839.
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