Download Distribution of lens sodium-potassium-adenosine

Distribution of Lens Sodium-PotassiumAdenosine Triphosphatase
Nicholas A. Delamere and William L. Dean*
Purpose. The specific activity of sodium-potassium-adenosine triphosphatase (Na-K-ATPase)
in lens fiber cells is lower than the specific activity in lens epithelium. To test whether there is a
reduction in the expression of Na-K-ATPase molecules in lens fibers, a Western blot technique
was used.
Methods. Membrane material was isolated from different regions of the rabbit lens. Na-K-ATPase (adenosine triphosphate hydrolysis) activity was measured in each membrane sample and
Western blots were performed using an antibody to rabbit kidney Na-K-ATPase.
Results. By immunoblotting, Na-K-ATPase polypeptide was detected in all lens cells. In contrast, adenosine triphosphate hydrolysis by the Na-K-ATPase (Na-K-ATPase activity) was not
detectable or was detectable only at very low levels in fiber membranes from the lens nucleus
and in cortex.
Conclusion. These findings suggest that plasma membrane adenosine triphosphatase enzyme
responsible for sodium-poiassium transport is expressed in newly formed lens fibers and the
transport molecules are retained as the fibers age and are compressed toward the center of the
lens. However, with fiber aging there is a loss of functional ability of the Na-K-ATPase to
hydrolyze adenosine triphosphate. Invest Ophihalmol Vis Sci 1 993;34:2159—2163.
liilectxolyte balance is vital to lens transparency.
Transparent mammalian lenses (including human
lenses) contain approximately 17 mmol/1 sodium and
130 mmol/1 potassium, whereas most senile cataracts
with opacification of the cortex have a dramatically
elevated sodium level (up to 100 mmol/1) and a potassium level that is well below 80 mmol/1 (see Patmore
and Duncan 1 for review). When lens sodium levels become abnormally high and lens potassium levels fall,
water accumulation causes cell disruption and impairment of transparency.
l-rmn Kentucky Liom Eye Research Institute and * University of Louisville School of
Medicine, Umisville, Kentucky.
Supported by USPHS Research Grant No. EY069I6, the Kentucky Eye Foundation,
and an unrestricted grant from Research to Prevent Blindness, Inc.
Submitted for publication April 22, 1992; accepted September 9, 1992.
Proprietary Interest: N.
Reprint requests: Nicholas A. Delamere, Department of Ophthalmology/Visual
Sciences, University of Louisville School of Medicine, Louisville, KY 40292.
It has long been established that the lens
contains sodium-potassium-adenosine triphosphatase
(Na-K-ATPase), and that this sodium pump mechanism is responsible for maintaining the electrolyte balance in the organ.2'3 Initially, Bonting and his coworkers2'3 and Palva and Palkama4 reported that the
Na-K-ATPase activity is localized almost entirely in the
lens epithelium. However, subsequent investigators
using more sensitive enzyme assay methods5"8 and cytochemical techniques9 have shown that there is measurable Na-K-ATPase activity in the lens fiber mass
even though the specific activity of Na-K-ATPase in
the epithelium is considerably higher than the specific
activity in fiber cells. Nevertheless, it should be remembered that although the fibers have a low specific
activity of Na-K-ATPase, these cells account for most
of the lens bulk and it turns out that the fiber mass
contains a significant fraction of the total Na-KATPase activity detectable in the lens.5"8
Investigative Ophthalmology & Visual Science.June 1993, Vol. 34, No. 7
Copyright © Association for Research in Vision and Ophthalmology
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Investigative Ophthalmology & Visual Science, June 1993, Vol. 34, No. 7
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The low specific activity of Na-K-ATPase in lens
fiber cells may signify that when the epithelium differentiates to become a fiber, there is a significant reduction in the relative expression of Na-K-ATPase molecules in the elongating cells, or that the Na-K-ATPase
in lens fibers becomes inactivated. In the current
study, we used immunoblotting to detect Na-K-ATPase polypeptides in different regions of the lens. Na-KATPase was detected in all lens cells, including cells in
the nucleus where ATP hydrolysis by the Na-K-ATPase
could not be measured.
MATERIAL AND METHODS
Lens Dissection
Lenses were obtained from 2-3 kg adult New Zealand
white rabbits immediately after they were killed with
an overdose of sodium pentobarbital administered by
a marginal ear vein. The procedures used in these studies were approved by the University of Louisville Institutional Animal Care and Use Committee and conformed to the ARVO Resolution on the Use of Animals in Research. Lenses were removed quickly from
the eyes by opening the posterior of the globe and
gently cutting the zonules. Using fine forceps, the capsule/epithelium was removed from the fiber mass.
Epithelium membranes and fiber membranes
from different sections of the lens were examined separately in these studies. After removal of the capsule/
epithelium, the lens fiber mass was frozen rapidly in
ep-
1
2
3
liquid nitrogen. Using a motor-driven trephine, a 6
mm diameter cylindrical pole-pole core was removed
from the frozen lens. The equatorial cortex region
surrounding the core was collected. The frozen polepole core was then divided into six anterior-posterior
sections as shown in Figure la.
Membrane Preparation
Using capsule/epithelium or sections of cortex, membranes were isolated by homogenization in ice-cold
buffer A (150 mmol/1 sucrose, 10 mmol/1 N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 1 mmol/1
dithiothreitol) containing the protease inhibitors leupeptin (10 fig/ml), PMSF (40 nM), aprotinin (0.03
trypsin inhibitor units/ml), antipain (10 /u.g/ml), and
pepstatin A (10 fjg/ml). The homogenate was centrifugated at 600 X g; this low-speed pellet containing debris and nuclei was discarded. The supernatant was
then placed in a centrifuge for 45 minutes at 100,000
X g. The pellet was resuspended in buffer A containing 0.6 M KC1 to remove extrinsic proteins and then
placed in a centrifuge again at 100,000 X g for 45
minutes. The pellet was then resuspended in buffer A,
placed in a centrifuge once more at 100,000 X g for 45
minutes to give a final pellet of partially purified membrane material that was resuspended in a small amount
of buffer A and stored in liquid nitrogen. The protein
content of the partially purified membrane material
was determined using the Peterson10 modification of
the Lowry assay."
5 6 equ
std
ep
FIGURE 1. (A) After removal of the capsule/epithelium, a 6 mm diameter cylindrical core was
removed from the lens. The core was divided into six anterior-posterior slices. The lens
equator material surrounding the core was also collected. (B) Immunoblot of rabbit lens
Na,K-ATPase polypeptides in membrane material isolated from epithelium (ep+), equatorial
fibers (equ) and fibers in six pole-pole sections (1 = Anterior. . . . 6 = Posterior).
Na,K-ATPase a subunit shows densely at s= 100 kDa; beta less clearly at « 4 0 kDa. In epithelial membranes prepared without protease inhibitors (ep—), loss of a subunit and appearance
of a Na,K-ATPase proteolysis product at = 3 0 kDa was observed. To obtain these results,
proteins were blotted to immobilon P membranes and probed with goat anti-(rabbit kidney
Na,K-ATPase). Immunopositive polypeptides were seen with alkaline phosphatase-coupled
antigoat gamma globulin. The molecular weight indications (100 k and 40 k) were determined from molecular weight standards in parallel lanes. (C) The polypeptide composition of
lens membrane material in these samples is shown in a Coomassie Blue-stained sodium dodecyl sulfate gel (C), which was run in parallel with the immunoblot.
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2161
Lens Na-K-ATPase
Measurements of Na-K-ATPase Activity
Na-K-ATPase activity was determined by adding membrane material to a buffer containing 100 mmol/1 sodium chloride, 10 mmol/1 potassium chloride, 40
mmol/l histidine, 3 mmol/1 magnesium chloride, 10~8
M calcium-ethyleneglycol-bis-(aminoethyl ether)N,N]tetraacetic acid buffer and 11 ng/m\ of alamethicin, at
pH 7.0 and 37°C. The alamethicin was added to make
all membrane vesicles accessible to the buffer constituents as described by Xie and coworkers.12 After 5 min
of preincubation, the ATPase reaction was started by
the addition of 1 mmol/1 ATP containing a tracer
amount of ATP labeled with 32P at the gamma position
(Amersham, Arlington Heights, IL). ATP hydrolysis,
which is linear up to 40 min, was quantified by determining the 32P liberated from labeled ATP using the
methods described by Socci and Delamere.13 To specify the ouabain-inhibitable Na-K-ATPase activity, the
reaction was run in the presence and absence of 10~3
M ouabain. Na-K-ATPase activity was expressed in
terms of protein (nmoles ATP hydrolyzed/hr//ug protein).
Immunoblotting
Membrane proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described by Laemmli14 on a 7.5% gel. Equivalent
amounts of membrane material (based on protein
content) were applied to each lane of the gel. The separated proteins were transferred to immobilon P membrane in 3-[cyclohexamino]-2-hydroxy-l-propanesulfonic acid buffer, pH 11.0 at 0.4 amps for 30 min. The
immobilon membrane was then blocked with 3% gelatin in TTBS (0.02 M Tris buffer at pH 7.5, 0.5 M
sodium chloride, and 0.05% Tween 20) and exposed
overnight to the primary Na-K-ATPase antibody (goat
anti-(rabbit kidney Na-K-ATPase), Calbiochem, San
Diego, CA). After washing the immobilon membrane
with TTBS, second antibody coupled to alkaline phosphatase was added for 1 hr. The immobilon membrane
was then washed again with TTBS before visualization of the Na-K-ATPase polypeptide immunoblots
with alkaline phosphatase substrates (Bio-Rad,
Richmond, CA).
RESULTS
Immunoblotting of Lens Na-K-ATPase
Membrane proteins were isolated from rabbit lens epithelium and from fiber membranes collected from different regions of the lens cortex and nucleus as described in Methods (Fig. la). Membrane proteins were
separated on 7.5% sodium dodecyl sulfate-polyacrylamide gels, transferred to immobilon membranes and
probed with an antibody raised against rabbit kidney
Na-K-ATPase. Na-K-ATPase polypeptides were detected in membrane material from all sections of the
lens; highest in the epithelium and lowest in fiber
membranes from the nucleus (Fig. lb). The Na-K-ATPase a subunit was clearly visible at around 100 kDa
and the /3 subunit less clearly at approximately 40 kDa.
The difference in detectability of the a and j3 subunits
may be a property of this particular antiserum. However, the /3 subunit was seen more clearly on the wet
immobilon membrane directly after color development of immunoblot, but the color faded during the
drying period.
In parallel with the immunoblot, another sodium
dodecyl sulfate gel was run and then stained with Coomassie Blue to reveal the polypeptide composition of
each lens membrane preparation (Fig. lc). Although
there was not a major polypeptide at 100 kDa, the
position of the Na-K-ATPase a subunit in the immunoblot, a higher molecular weight (116 kDa) major protein band appeared in samples from lens fiber, but not
in the epithelium. It is therefore unlikely that spurious, nonspecific binding of Na-K-ATPase antibodies
to major lens proteins contributed to our findings. In
addition, the stained gel illustrates that there was little
contamination of the epithelial membrane preparation with fiber material since the major 116 kDa fiber
protein band is nearly absent from the epithelial preparation.
To obtain the membrane preparations used in
these studies, lens material was homogenized and centrifugated at cold temperatures in the presence of
protease inhibitors. To test whether the Na-K-ATPase
is sensitive to proteolytic breakdown, a sample of epithelium was prepared in the absence of protease inhibitors and the sample (sample ep-, Fig. lb) was allowed
to come briefly to room temperature. In an immunoblot of this membrane preparation there was evidence
of considerable conversion of the Na-K-ATPase a and
possibly j8 subunits to a small immunoreactive polypeptide appearing significantly below 40 kDa.
Na-K-ATPase Activity
The Na-K-ATPase activity (rate of ouabain-sensitive
ATP hydrolysis) was measured in each of the membrane samples isolated from lens epithelium or various
regions of the lens fiber mass. The highest Na-K-ATPase activity was observed in the epithelium, which
contained 50% of the total lens Na-K-ATPase (Table
1). However, it should be stressed that detectable levels of Na-K-ATPase activity were observed in fiber
membranes isolated from the deeper cortex (Sections
2 and 5). Na-K-ATPase activity was not detectible in
material from the nucleus (Sections 3 and 4). These
same sections contained substantial amounts of Na-KATPase a subunit as indicated by immunoblotting
(Fig. lb).
It is important to note that the equatorial section
of the fiber mass contained approximately 32% of the
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Investigative Ophthalmology & Visual Science, June 1993, Vol. 34, No. 7
2162
TABLE l. Na,K-ATPase Activity Determined
in Sections of the Rabbit Lens
Lens
Section
Epithelium
Anterior 1
2
3
4
5
Posterior 6
Equatorial
Specific Activity
of Na,K-ATPase
(nmol ATP/figprotein/hr)
% of Total Lens
Na,KATPase
Activity
6420 (373)
242 (27)
53 (10)
11 (24)
20(11)
118 (26)
278 (37)
1226(92)
50.9
4.7
1.4
0.4
0.7
4.1
5.4
32.3
Data are die mean ± SE of six measurements. Lenses were dissected into sections as shown in Figure 1 A. Na,K-ATPase activity
was determined in membrane material isolated from each section.
total lens Na-K-ATPase activity. This section of the
lens is made up mostly of fiber "middles"; the tips of
each lens fiber extend toward the sutures at the poles
of the lens.
DISCUSSION
Na-K-ATPase is found in the plasma membranes of
cells as a heterotetrainer of 100 kDa a subunits and 40
kDa /? subunits.15'16 By immunoblotting, we were able
to detect the Na-K-ATPase a subunit very clearly in
lens membranes. The Na-K-ATPase /? subunit was less
demonstrable, perhaps because of technical difficulties; the jS subunit band faded as the immobilon membrane dried.
The key feature of this study is that we observed
regions of the lens that have considerable amounts of
Na-K-ATPase polypeptides but exhibit very low or negligible ouabain-sensitive ATP hydrolysis activity (Na-KATPase activity). In the lens nucleus, for example, the
Na-K-ATPase activity was nil, although the fiber cell
membranes in that region react with the Na-K-ATPase
antibody. This finding suggests that as lens epithelium
differentiates to give rise to fiber cells, Na-K-ATPase is
expressed in the newly formed fibers and the transport
molecule is retained by these cells as they age and are
compressed toward the center of the lens. However,
our data indicate that with fiber differentiation and
subsequent aging, there is a loss in the functional ability of the Na-K-ATPase to hydrolyze ATP. For example, membrane samples from lens epithelium had approximately 25 times more Na-K-ATPase activity than
superficial cortical fibers at the anterior or posterior
pole. Yet all these regions had a relatively similar NaK-ATPase immunoblot reaction. Presumably, the fiber
cell Na-K-ATPase that cannot hydrolyze ATP is also
unable to transport sodium or potassium.
The Na-K-ATPase was susceptible to proteolysis.
Loss of a and fi subunits of Na-K-ATPase was observed
in epithelial membranes prepared without protease inhibitors (Fig. lb); in this membrane sample, a small
(less than 40 kDa) immunoreactive polypeptide was
observed, perhaps representing Na-K-ATPase breakdown products. There was no evidence of such lowmolecular-weight Na-K-ATPase breakdown products
in membrane material isolated in the normal manner.
The lack of Na-K-ATPase breakdown fragments in
fiber membranes from lens nucleus suggests that there
may be relatively little proteolysis of lens Na-K-ATPase
as fiber cells age.
On the basis of this study, we can only speculate
on the possible reasons why lens fiber Na-K-ATPase
appears to lose functional activity as it ages. Lens fiber
membranes have an unusually high cholesterol/phospholipid ratio.17 The cholesterol/phospholipid ratio,
which increases toward the center of the lens, gives
lens fiber membranes an unusually high degree of
order; the membranes are rigid.18 It is possible that the
lack of membrane fluidity impairs the functional ability of fiber cell Na-K-ATPase, particularly in the nucleus where fluidity is lowest. An influence of membrane fluidity on Na-K-ATPase activity has been observed in a number of cell types.19'20 Low membrane
fluidity might also inhibit the activity of plasma membrane Ca-ATPase in lens fibers as suggested by the
temperature-dependence studies of Delamere and coworkers.21
If lens fiber Na-K-ATPase indeed becomes inactivated as the fiber cells age, lens sodium—potassium balance must rely heavily on a functional Na-K-ATPase in
the epithelium and equatorial cortex; together, these
two regions account for more than 80% of the ouabain-sensitive ATP hydrolysis activity in the lens (Table
1). The specific activity of Na-K-ATPase in the equatorial region of the fiber mass was considerably higher
than that in the fiber cell samples collected from the
anterior-posterior core, which we removed from the
optical axis of the lens. Each lens fiber stretches from
the anterior to the posterior pole; our observations
suggest that the tips of lens fibers contain less functional Na-K-ATPase molecules than the middles (equatorial region) of the cells. It is possible that active sodium-potassium transport by fiber cell Na-K-ATPase
at the equator plays an important role in lens electrolyte regulation. A role for the equatorial portions of
fibers in performing ion transport duties for the whole
lens is consistent with reports that extensive gap junctions between adjacent fibers are seen near the lens
equator and not close to fiber tips.22-23 Ion pumping by
cells close to the surface can perhaps influence the
cytoplasmic composition of underlying packed cells in
regions where cell-cell coupling by gap junctions is
extensive. Additionally, extracellular diffusion path-
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2163
Lens Na-K-ATPase
ways that could channel sodium out from the lens are
best developed at the lens equator24 and the lens equator is associated with a marked outward potassium
current.25
12.
Key Words
13.
Na-K-ATPase, lens, rabbit, immunoblot, Ca-ATPase
14.
References
15.
1. Patmore L, Duncan G. The physiology of lens membranes. In: Duncan G, ed. Mechanisms of Cataract Formation in the Human Lens. London: Academic Press;
1981:193-217.
2. Bonting SL, Caravaggio LL, Hawkins NM. Studies on
sodium-poiassium-activated adenosine triphosphatases. VI. Its role in cation transport in the lens of cat,
calf and rabbit. Arch Biochem Biophys. 1963; 101:4755.
3. Bonting SL. Na-K activated adenosine triphosphatase
and active cation transport in the lens. Invest Ophthalmol Vis Sci. 1965;4:723-738.
4. Palva M, Palkama A. Electronmicroscopical, histochemical and biochemical findings on the Na,K-ATPase activity in the epithelium of the rat lens. Exp Eye
Res. 1976; 22:229-236.
5. Fournier DJ, Patterson JW. Variations in ATPase activity in the development of experimental cataracts. Pro
Soc Exp Biol Med. 1971; 137:826-832.
6. Neville MC, Paterson CA, Hamilton PM. Evidence for
two sodium pumps in the crystallin lens of the rabbit
eye. Exp Eye Res. 1978;27:637-648.
7. Kobayashi S, Roy D, Spector A. Sodium/potassium
ATPase in normal and cataractous human lenses. Curr
Eye Res. 1983;2:327-334.
8. Alvarez LF, Candia OA, Grillone LR. Na+-K+ATPase
distribution in frog and bovine lenses. Curr Eye Res.
1985;4:143-152.
9. Unakar NJ, Tsui JY. Sodium-potassium-dependent
ATPase I. Cytochemical localization in normal and cataractous rat lenses. Invest Ophthalmol Vis Sci.
1980;19:630-641.
10. Peterson GL. A simplification of the protein assay
method of Lowry et al which is more generally applicable. Ann Biochem. 1977;83:346-356.
11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Protein measurement with the folin phenol reagent, f
BiolChem. 1951; 193:265-275.
Xie Z, Wang Y, Ganjeizadah J, McGee R, Askari A.
Determination of total (Na+-K+)-ATPase activity of
isolated or cultured cells. Anal, Biochem. 1989;
183:215-219.
Socci RR, Delamere NA. The effect of vanadate upon
calcium-stimulated ATPase of the rabbit iris-ciliary
body. Invest Ophthalmol Vis Sci. 1988;29:1866-1870.
Laemmli UK. Synthesis of T4 phage proteins. Nature.
1970;217:680-685.
Peterson GL, Hokin LE. Molecular weight and stoichiometry of Na,K-ATPase subunits. J Biol Chem.
1981; 256:3751-3761.
Geering K. Subunit assembly and functional maturation of Na,K-ATPase. J Membrane Biol. 1990;
115:109-121.
Borchman D, Delamere NA, McCauley LA, Paterson
CA. Studies on the distribution of cholesterol, phospholipid, and protein in the human and bovine lens.
Lens Eye Toxic Res. 1989;6(4):703-724.
Borchman D, Herrell P, Yappert MC. Structural characterization of human lens membrane lipid by infrared speclroscopy. Invest Ophthalmol Vis Sci.
1991;32:236-248.
Sinensky M, Pinkerton F, Sutherland E, Simon FR.
Rate limitation of Na,K-ATPase by membrane acyl
chain ordering. Proc Nail Acad Sci USA. 1979;
76:4893-4897.
Yeagle PL, Young J, Rice D. Effects of cholesterol on
Na,K-ATPase ATP hydrolyzing ability in bovine kidney. Biochemistry. 1988; 27:6449-6452.
Delamere NA, Paterson CA, Borchman D, King KL,
Cawood SC. Calcium transport, Ca-ATPase and lipid
order in rabbit ocular lens membranes. AmfPhysiol.
1991a;260:C731-C737.
Brown HG, Kahn A, Kuszak JR. The polar arrangement of imps and gap junctions in lens fiber cells. Invest Ophthalmol Vis Sci. 1988;29(suppl):428.
Kuszak JR, Brown HG, Peterson KL. Electron microscopic observations of the crystallin lens. J Electron Mic
Tech. (in press).
Lo W-K. In vivo and in vitro observations on permeability and diffusion pathways of tracers in rat and
frog lenses. Exp Eye Res. 1987;45:393-406.
Wind BE, Walsh S, Patterson JW. Equatorial potassium currents in lenses. Exp Eye Res. 1988; 46:117—
130.
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