Lens Epithelium and Fiber Na,K-ATPases: Distribution and Localization by Immunocytochemistry

Abstract

purpose. To use immunofluorescence and immunogold techniques to identify the catalytic subunits of the Na,K-ATPases of the lens and to determine their location in the cells of the epithelium and cortex of bovine and human lenses.

methods. Frozen sections of capsulated and decapsulated bovine and human lenses were prepared, blocked, and treated with affinity-purified polyclonal rabbit antibodies to the Na,K-ATPase catalytic subunit isoforms with subsequent treatment with fluorescein isothiocyanate–labeled goat anti-rabbit IgG and visualization of the fluorescence by light microscopy. An immunogold-labeled goat anti-rabbit IgG was used to detect, by electron microscopy, the binding of the same affinity-purified polyclonal antibodies to thin sections of decapsulated lenses that had been fixed and embedded in Lowicryl K4M. The results were confirmed by staining of western blot analysis of sodium dodecyl sulfate–polyacrylamide gel separations of enriched membrane preparations from bovine and human lenses.

results. The three common catalytic subunits of the Na,K-ATPases are present in the plasma membranes of lens epithelium, lens fibers, or both. The data indicate a polarized distribution of the α1 and α3 catalytic subunit isoforms in central epithelium. In the cortical fibers, theα 2 isoform is present around the interdigitations. The α3 isoform is found in the interdigitation-free regions of human cortical fibers.

conclusions. This unique distribution of Na,K-ATPases precludes the popular pump-leak model for lens monovalent cation homeostasis. The functional significance of the distribution of Na,K-ATPases in the lens epithelium and superficial fibers is currently under investigation.

In many cataractous lenses, Na⁺ concentrations are elevated and K⁺ concentrations are lower than normal. ^{1 2} The causes of the monovalent cation imbalances remain elusive. The most popular theories involve increased plasma membrane permeability with or without cation transporter inhibition. There is evidence that the Na,K-ATPases (monovalent cation transporters) of the lens epithelium of cataractous human lenses are inhibited or that they display unusual steady-state ATP hydrolysis kinetics. ^{3 4} There is equally compelling evidence to suggest that K⁺ transport is normal and that membrane permeability is increased. ^{5 6 7}  

Historically, the lens has been viewed as a syncytium ^{8 9 10} in which the lens epithelium is the site of Na,K-ATPase–dependent Na⁺/K⁺ exchange. The lens fibers, on the other hand, are believed to be permeable to Na⁺ and K⁺. This simple model explains, quite adequately, the K⁺ current (anterior to posterior) and the Na⁺ current (posterior to anterior) in normal lenses. ¹¹ More recently, data have been reported to suggest that the lens fiber cells are rather impermeable and use their own Na,K-ATPases to maintain monovalent cation homeostasis. ^{12 13} Furthermore, there are two Na⁺ currents, the one from anterior to posterior and one at the lens equator. ^{14 15}  

The question then arises as to the mechanism by which relatively impermeable fiber cells and the metabolically active lens epithelium interact to maintain ion homeostasis. To answer this question, the location and mechanism of action of the major lens ion transporters and carriers need to be described for normal clear lenses. Our focus has been the lens Na,K-ATPases. ^{12 13} In this report, we use immunocytochemistry to describe the distribution of Na,K-ATPase catalytic subunits in the plasma membranes of bovine lenses and clear human lenses. 

Materials and Methods

Tissues

Bovine eyes, purchased from a local abattoir within 3 hours of death, and human eyes, obtained within 12 hours of death from the Fort Worth Eye Bank, were processed immediately. 

Catalytic subunit-specific polyclonal peptide antisera, to extracellular (EC) and intracellular (IC) epitopes of the three catalytic subunit isoforms were prepared by injection of KLH-peptide complexes into New Zealand white Rabbits. The peptides used to make the antisera were MGKGVGRDKYEPAAVSEHGDKK for α1-IC, MGRGAGREYSPAATTAENGGGK for α2-IC, MGDKKDDKSSPKK for α3-IC, GIRSATEEEPPNDDLYK for α1-EC, GIKAAMEDEPSNDNLYK for α2-EC, and GIQAGTEDDPSGDNLYK for α3-EC. The peptides were prepared with an Applied Biosystems Peptide Synthesizer at the UCLA Molecular Biology Institute Peptide Synthesis Facility (model 430A). The peptide sequences were confirmed by solid-phase sequence analysis (Commonwealth Biotechnologies, Richmond, VA). 

The resultant antisera were affinity purified. For the preparation of each affinity resin, 4 ml of Affigel-Hz was reacted for 2 hours with 10 ml of 12.5% glutaraldehyde in 1× Affigel-Hz coupling buffer (supplied by Bio-Rad, Hercules, CA). After the coupling of glutaraldehyde, the resin was washed 2 times with deionized water and 5 times with 100 mM phosphate buffer (pH 6.0). Ten milliliters of peptide solution (0.5 mg/mL of the specific peptide in 100 mM phosphate buffer, pH 6.0) and 2 mg of NaCNBH₃ were added to the washed resin and mixed overnight. The phosphate buffer was removed; the resin was washed with 50 ml of 100 mM NaHCO₃ and reacted for 30 minutes with an additional 3 mg of NaCNBH₃ in the sodium bicarbonate buffer. The NaCNBH₃ was required to reduce the Schiff base formed between the aldehyde of the resin and the amine of the peptide or peptides. The resin was rinsed with 100 mM Na₂CO₃ (20 ml) followed by 50 ml of deionized water. The resin was stored in 0.5 mM NaN₃. 

For affinity purification, the affinity resins were transferred to Poly-Prep columns (Bio-Rad) and rinsed with 0.02 M Tris buffer (TBST), pH 7.5, containing 0.150 M NaCl and 0.15% Tween-20 (Bio-Rad). The resin (approximately 2 ml, packed) was mixed with antiserum (0.5 ml diluted with 1.5 ml of TBST) and placed on a shaker for 2 hours. The column was then rinsed with 20 ml of TBST. Each TBST eluate was saved. Each resin was then incubated for 1 minute in 0.1 M glycine (Gly) buffer, pH 2.8. Each Gly eluate was collected in tubes containing 41μ l of 1 M Tris buffer, pH 9.5. Each resin was incubated for 1 minute with an additional 1 ml of Gly buffer. These eluates were collected in tubes containing 41 μl of 1 M Tris buffer and combined with the previous Gly eluates. Sufficient bovine serum albumin and NaN₃ were added to the TBST and Gly eluates to yield a final concentration of 1% and 0.1 mM, respectively. The TBST and Gly eluents were aliquoted and stored at −70°C. 

The effectiveness of the affinity purification was tested by slot blot analysis using rat brain microsomes (1 mg/mL) as the antigen. For the slot blot analyses, the immune complexes were visualized using horseradish-peroxidase–labeled goat anti-rabbit IgG (hrp-GARIgG; Boehringer–Mannheim, Indianapolis, IN) and the hrp-substrate, 4-chloro-1-napthol. 

To test the specificity of the affinity purified antibodies, western blot analysis of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) separations of microsomal fractions from rat brain, rat heart, and rat kidney were stained with each of the six antibodies. Microsomal fractions were prepared using previously described protocols. ^{4 12 13 16 17 18 19 20 21} Visualization was with hrp-GARIgG with 4-chloro-1-napthol as the hrp-substrate. Interspecies cross-reactivity was checked using rat, canine, and bovine microsomal preparations. 

Immunofluorescence staining was performed on frozen sections. Bovine and human lenses were cut into quarters and fixed with 4% paraformaldehyde. After sequential immersion in 10%, 15%, and 20% sucrose solutions, the lens quarters were embedded with OCT compound in liquid nitrogen. Cryosections at 6 μm, prepared with a cryostat, were collected on glass slides and stained. Before staining, the sections were blocked with normal goat serum for 1 hour before incubation with the primary antisera, followed by incubation with fluorescein isothiocyanate–labeled GARIgG (FITC-GARIgG). For the negative controls, sections were incubated with normal rabbit serum or the appropriate TBST eluate, followed by incubation with FITC-GARIgG. A Nikon Diaphot light microscope (40× objective) was used to visualize the stained sections. Photomicrographs were collected as permanent records of the immunofluorescence results. Exposure times of 15 seconds were used for preparations treated with the affinity-purified antisera (Gly fractions). Exposure times of 30 seconds were used for the samples treated with the TBST fractions from the affinity purification of the antisera and for the normal rabbit serum controls. 

Immunoelectronmicroscopy was performed on isolated lens fiber plasma membrane preparations as well as on fixed bovine and human lenses. To prepare fractions enriched in fiber cell plasma membranes from bovine and human lenses, the following protocol was used. After the lens capsule epithelium was removed, the lens cortex was collected as described previously. ^{12 13 21} The cortex was homogenized in ice-cold 0.5 M Tris buffer, pH 8.0. The homogenate was centrifuged at 7000 rpm for 15 minutes. The supernatant of the 7000 rpm centrifugation was centrifuged at 25,000 rpm (Beckman Ti45 rotor) for 20 minutes. The resultant pellet was washed twice with ice-cold 0.5 M Tris buffer. A microsomal fraction from the bovine renal medulla was prepared using previously described procedures. ¹⁷ The lens and renal membrane preparations were fixed (4% paraformaldehyde/0.1% glutaraldehyde) for 1 hour. The fixed membranes were embedded in Lowicryl K4M. Thin sections at 0.1 μm, on grids, were blocked for 1 hour in 50 mM Tris buffer, pH 7.5, containing 0.05% Triton X-100 and bovine serum albumin (5 mg/ml). The grids were incubated overnight at 4°C with the appropriate primary antisera, rinsed, and incubated with GARIgG coupled to 15 nm gold particles for 1 hour. After rinsing, the sections were post-fixed with 2% glutaraldehyde and stained with uranyl acetate and lead citrate. For the studies with the human lens, the quartered lens was fixed in 4% paraformaldehyde/0.1% glutaraldehyde and embedded in Lowicryl K4M. After removal of the nucleus from bovine lens quarters, the remaining cortex was fixed in 4% paraformaldehyde/0.1% glutaraldehyde and embedded in Lowicryl K4M. The procedure for preparation of and staining of the thin sections on grids was identical with that described previously for the lens fiber cell membranes. A Zeiss 910 transmission electron microscope was used to visualize the stained sections. 

Variation in immunogold-labeling patterns was determined from counts of gold particles in multiple fields. For bovine lens fiber cell membrane preparations, gold particles were counted in 48 fields (4μ m² each) on 12 grids from two separate membrane preparations. For human lens fiber cell membranes, gold particles were counted in 35 fields (4 μm² each) on seven grids from a single lens fiber cell membrane preparation. The results for the membrane preparations are reported as particles/4 μm² field. For the bovine and human lens sections, the total length of the membrane in 24 fields (4μ m² each) was measured. The measured length of the membrane in interdigitations was subtracted from the total length of the membrane to determine the length of membrane devoid of interdigitations. Counted gold particles in the membrane are reported as particles per micrometer of membrane. The data are presented as mean ± SD. Using an unpaired Student’s t-test (Statveiw; Abbacus), the significant differences in immunogold labeling were determined. 

Results

Affinity Purification of the Six Antibodies

All three catalytic subunit isoforms are present in rat brain microsomes. The Gly fractions of the affinity purification of the six antisera stained rat brain microsome preparations on slot blots. There was no staining of slot blots of rat brain microsome preparations with the TBST fractions. For all succeeding experiments, the TBST fractions as well as normal rabbit serum were used as negative controls for Gly fractions, used for the western blot analysis and immunocytochemistry. 

Specificity of the Six Antisera

Western blot analysis of SDS–PAGE separations of brain, kidney, and heart microsome preparations was used to determine the specificity of the affinity-purified antisera. Rat, canine, and bovine microsome preparations were tested. The results for the rat preparations (Fig. 1) are representative of these studies. The data for the canine and bovine preparations are not included and will be presented in a future report of our studies with cultured lens epithelium. 

The affinity-purified antisera α1IC and α1EC stained major bands at∼ 95 kDa in all microsome preparations. This is in agreement with previous studies that have demonstrated the presence of the α1 isoform in kidney, heart, and brain. Lower-molecular-weight bands are also stained with the α1IC and α1EC antisera. ^{22 23 24 25 26} The apparent molecular weights of the lower-molecular-weight bands stained by the α1IC antiserum are 70 kDa (brain), 40 to 42 kDa (heart and kidney), and 35 to 37 kDa (heart and kidney). Of these, only the 35- to 37-kDa band is recognized with the α1EC antiserum. This band is probably a cleavage product of the α1 subunit. Previous studies have demonstrated that the α1 isoform is highly susceptible to proteolytic cleavage. ^{27 28 29 30} The larger bands (70 kDa and the 40–42 kDa) are not stained with the α1EC antiserum but are stained with the α1IC antiserum. The α1IC site is before the α1EC site, and both EC and IC sites are within the first 250 residues. Therefore, the 70-kDa and 40- to 42-kDa bands are not Na,K-ATPase degradation products but proteins that contain regions homologous to the sequence chosen for the α1IC antiserum. 

The α2IC and α2EC antisera stained prominent bands at 95 kDa in the heart and brain preparations. There were no bands stained in the kidney preparations. These results are in agreement with previous studies that demonstrated the absence of α2 in the kidney and the presence of α2 in the brain and heart. ^{22 23 25 26 31} There was faint staining of a 70-kDa band in the brain preparation by both the α2IC and α2EC antisera. This suggests limited proteolytic cleavage. 

The α3IC and α3EC antisera stained a 95-kDa band in the brain preparation. There was no staining evident in the kidney and heart preparations. These results are in agreement with those of previous studies that have demonstrated that the α3 isoform is found primarily in the central nervous system. ^{22 23 25 26 31}  

Localization by Indirect Immunofluorescence

The six antisera were used to determine the presence and location of the Na,K-ATPase catalytic subunits in the bovine lens by indirect immunofluorescence. The results for the three EC antisera (Fig. 2) suggest that all three catalytic subunits are present in the superficial regions of the bovine lens. The α1 isoform is present in the epithelium and in the superficial fibers. In the central region of the lens epithelium (Fig. 2A) the staining is heaviest on the basal and lateral surfaces. In the equatorial epithelium (Fig. 2E) staining of the α1 isoform is observed on the apical, basal, and lateral cell borders. In the superficial cortical fibers, the α1 isoform is observed as punctate spots on the fiber surface. There is little if any evidence of the α2 isoform in the central epithelium because the fluorescence intensity shown in Figure 2B is similar to that for the control (Fig. 2D) . There is heavy staining of the equatorial epithelium and superficial fibers with the α2EC antiserum. The α2 isoform is observed as punctate spots on the surface of the superficial fibers (Fig. 2F) . The α3 isoform would appear to be localized to the apical and lateral borders of cells in the central epithelium (Fig. 2C) . At the equator, the α3 isoform is present on apical, basal, and lateral surfaces of the epithelium and on the borders of the underlying fiber cells (Fig. 2G) . Staining of the lens epithelium and superficial cortical fibers was not observed with the three IC antisera (data not included). 

The results with the three EC antisera (Fig. 3) suggest that the catalytic subunits appear as patches on the fiber edge or in punctate patterns on the fiber cell surface in the cortex of decapsulated bovine and human lenses. In the deeper cortical fibers of the bovine lens (Figs. 3A 3D) , staining with antisera to the α2 andα 1 isoforms is observed. There is little if any evidence of staining with α3EC (Fig. 3G) in this region of the bovine lens. In the deeper fibers of the human lens (Figs. 3B 3H) , α2EC and α3EC stained the fibers. There is little if any evidence of staining with α1EC (Fig. 3B) in this region of the bovine lens. There was no evidence of staining with the three IC antisera in this region of the lens (data not included). 

Immunogold Labeling and Transmission Electron Microscopy

To better characterize the discontinuous localization of the α2 and α3 catalytic subunit isoforms in human fiber cells, sections from the superficial cortex of decapsulated human lenses were immunogold-labeled and studied by electron microscopy. Although the description of the immunogold-labeling results focuses on the quantitation of gold particles in multiple fields, representative micrographs for the α1EC, α2 EC, and α3EC antisera are shown in Figures 4 A, 4B, and 4C, respectively. There were 2 ± 3 gold particles/μm_membrane for all the TBST controls and for the α1EC antiserum. With the α2EC antiserum, there were 46 ± 16 particles/μm_membrane (P < 0.001 when compared with the TBST control) adjacent to or associated with interdigitations (see Fig. 4B as an example). In regions devoid of interdigitations, the number of gold particles was considerably lower (4 ± 3 particles/μm_membrane) and not significantly higher, statistically, than that of the controls (3 ± 2 particles/μm_membrane; Figs. 4E and 4F ). For theα 3EC antiserum, gold particles were not prevalent in the interdigitations (2 ± 2 particles/μm_membrane) but were present in regions devoid of interdigitations (10 ± 5 particles/μm; P < 0.001). Similar experiments carried out with the superficial cortex of decapsulated bovine lenses suggested α2 at the interdigitations (25 ± 12 particles/μm_membrane; P < 0.001), α1 in the regions relatively free of interdigitations (5 ± 2 particles/μm_membrane; P = 0.048), and the absence of α3 (2 ± 2 particles/μm_membrane; P > 0.1). Representative micrographs for the superficial bovine lens cortex have not been included. 

The positive results were with the antisera to the extracellular epitopes. As pointed out in the previous paragraphs, similar studies with the antisera to the intracellular epitopes were negative. The lack of success with the three IC antisera was most likely a problem with penetration of the antiserum, availability of the antigenic determinant(s) in the fixed tissue, or both. To circumvent this problem, membranes, isolated from the bovine renal medulla as well as bovine and human lens cortex, were fixed, immunogold-labeled, and studied by transmission electron microscopy. Representative micrographs for the α2IC and α2EC are shown in Figure 5 . The quantitative results for multiple fields were collected into Table 1 . Usually, at least one gold particle appeared in all fields studied. There were no statistically significant differences among the values for the TBST controls for the three membrane preparations. The values for the α1IC and α1EC Gly fractions for microsomes of the bovine renal medulla were significantly greater than those of the TBST controls, a result expected based on the immunoblot data (Fig. 1) . The values for the Gly fractions for the α2 and α3 epitopes were not significantly different from those of the TBST controls. For the human lens membrane preparations, the number of gold particles observed with the Gly fractions of the antisera to the α2 and α3 epitopes was significantly greater than those of their respective TBST controls. Furthermore, the number of gold particles with the α2IC and α2EC Gly fractions was significantly greater than the number observed with the α3IC and α3EC Gly fractions. For the bovine lens preparations, only the preparations stained with the α2IC and α2EC Gly fractions had significantly more gold particles than the respective TBST controls. This result was unexpected because the results with α1EC antiserum in the superficial bovine lens cortex suggested low levels ofα 1 in the membrane regions free of interdigitations. 

To confirm the immunocytochemistry, enriched membrane fractions, isolated from the bovine lens central epithelium, equatorial epithelium, cortex, and the decapsulated human lens were separated by SDS–PAGE, transferred to nitrocellulose, and treated with the antisera to the catalytic subunit isoforms (Fig. 6) . For the membranes from the bovine lens cortex, no bands were identified by the α1EC, α1IC, α3EC, or α3IC antisera (data not included). With the α2IC antiserum (Fig. 6 , lane c) a band, MW ∼90 kDa, was identified. The 90-kDa band and a 78-kDa band were identified with the α2EC antiserum (Fig. 6 , lane d). This suggests cleavage of a 10- to 12-kDa fragment from the amino terminus because the 78-kDa band was not apparent when the blot was stained with the α2IC antiserum. For the membranes (a microsomal fraction) from the central epithelium of the bovine lens, a 90-kDa band was identified by the α1IC (Fig. 6 , lane e) and the α3IC (Fig. 6 , lane k) antisera; no bands were identified with the α2IC antiserum (Fig. 6 , lane h). For the membranes (a microsomal fraction) from the equatorial epithelium of the bovine lens, a 90-kDa band was identified by the α1IC (Fig. 6 , lane f) and α2IC (Fig. 6 , lane i) antisera; lower-molecular-weight bands were identified by the α1IC and α3IC antisera (Fig. 6 , lanes f and l). The α2IC antiserum identified a 90- to 95-kDa band from the SDS–PAGE separation of the water-insoluble fraction ³² of a decapsulated human lens. The human lens was obtained within 6 hours of death. Studies with other human lenses indicated significant degradation of the Na,K-ATPase catalytic subunit of the lens fiber cells to molecular weight ≥10 kDa when lenses were received at times≥ 10 hours postmortem. Furthermore, with the significant loss of cells from the capsule epithelium of the postmortem human lenses, immunocytochemical and immunoblot analysis results for the distribution of the Na,K-ATPase catalytic subunits were mixed and, therefore, have not been included in this report. 

Discussion

The results with the IC panel of antisera and the denatured lens preparations suggest that α1 and α3 are the predominant isoforms in the bovine lens epithelium and that α2 is the predominant form in the bovine and human lens fibers. This result was confirmed by immunocytochemistry with the EC panel. Immunocytochemistry results with the IC panel were negative. Differential antibody penetration is a plausible explanation for the disparate immunofluorescence results with the two antibody panels. The epitopes, recognized by the three EC antisera are extracellular. ^{33 34 35} The epitopes for the IC antisera, on the other hand, are intracellular. ³³ Permeabilization of the lens sections with detergents or methanol, which was not part of our experimental protocol, might have allowed adequate antibody penetration to the site recognized by the IC antisera. However, such treatments would be expected to compromise the plasma membrane. 

Even if permeabilization was not an issue, negative results might be expected with the three IC antisera. The EC domain of Na,K-ATPase catalytic subunits, proposed from analysis of hydropathy plots and predicted secondary structure, consists of relatively short segments of polar and/or charged amino acids. ^{36 37} It would be difficult to envision that these short segments are so buried in the tertiary structure that they are inaccessible to solvent, solutes, and antibodies. On the other hand, the IC domain of the Na,K-ATPase catalytic subunits consists of several long segments of sequence, the N-terminal (∼90 amino acid residues), ^{33 38 39} the region between membrane spanning sequences 2 and 3 (∼125 amino acid residues), ³³ and the large region between membrane spanning sequences 4 and 5 (∼480–500 amino acid residues). ^{40 41 42} The hydrophilic N-terminal epitopes, targeted by the three IC antisera, may be buried in the tertiary structure, the quarternary structure, or both of the native enzyme, thus unreactive. Alternatively, the antigenic determinants (epitopes) may be structurally altered due to kinase-dependent phosphorylation ^{38 43 44 45} or inaccessible due to the interacting cytoskeleton. ^{42 46 47 48} Although future studies will address the accessibility of the three IC epitopes in the native structure, the results shown in Figures 1 and 6 clearly demonstrate positive staining of a 90-kDa band by all six antisera when the catalytic subunits are denatured and separated by SDS–PAGE. 

To date, the only reported differences in the mechanism of action of Na,K-ATPases containing the α1, α2, or α3 catalytic subunit isoforms are the IC₅₀ for Na⁺ inhibition of ATP hydrolysis (1.15 ± 0.13, 1.05 ± 0.11, and 3.08 ± 0.06 mM, respectively, forα 1, α2, and α3) and the relative affinities for ATP (0.43 ± 0.12, 0.54 ± 0.15, and 0.21 ± 0.04 mM, respectively, forα 1, α2, and α3). ³⁴ A second question arose from this observation, and a review of the results is presented in this report. What is the purpose of multiple Na,K-ATPases in the same cell? 

In the central epithelium of the bovine lens, both α1 and α3 appear to be expressed and to be localized in the plasma membrane with theα 1 basal and lateral and the α3 apical and lateral. Chondrocytes of the articular cartilage and neurons are the only other cells for which immunocytochemistry studies have demonstrated the presence of the α1 and α3 catalytic subunit isoforms in the same cell. ^{24 49} Chondrocytes exist in a unique environment in which free EC Na⁺ levels are 250 to 400 mM because of the high density of fixed negative charges on the glycosaminoglycans in the EC matrix. ⁴⁹ The lens capsule has a high glycosaminoglycan content, which may contribute to elevated EC Na⁺ levels at the apical surface of the lens epithelium. Whether elevated EC Na⁺ induces α3 catalytic subunit expression remains to be determined. Similar to the results with the cells of the central epithelium, the expression of the α1 and α3 isoforms is polarized in neurons with the α1 predominant in the dendrite and α3 predominant in the axon. ²⁴ It should be pointed out that the EC matrix of the central nervous system is extensive and complex. ⁵⁰ Glycosaminoglycans are involved in central nervous system development and have been implicated as players in the etiology of Alzheimer’s disease. ^{51 52 53 54} In fact, the adhesion molecule on glia (AMOG) protein, a cell surface component involved in EC matrix interactions in the brain, is one of the three possible glycoprotein subunit isoforms of the Na,K-ATPase. ^{55 56 57 58} Studies currently in progress will address and clarify the issue of EC matrix interactions with the Na,K-ATPases of the cells of the central epithelium of the bovine lens. Other studies currently in progress in our laboratory will address the role of the cytoskeleton in the polarization of the α1 and α3 catalytic subunits in the central epithelium of the bovine lens. 

In the equatorial epithelium, there is an absence of α3/α1 polarization and the appearance of the α2 isoform, a result that confirms previous studies of rat lens epithelium. ^{13 59} Although the importance of the changes in catalytic subunit distribution in fibrogenesis remains to be elucidated, increased IC Na⁺ would appear to be the signal for induction of the α2 subunit. Both an amphotericin-induced increase in membrane permeability and cardiac glycoside inhibition of Na,K-ATPase have been shown to induce the expression of the α2 subunit isoform. ^{60 61}  

In the cortical lens fibers of bovine and human lenses, α2 is predominant. The α2 is a constituent of, or adjacent to, interdigitations between cells. The α3 (human) isoform and perhaps the α1 (bovine) isoform, which are present in much lower concentrations than the α2 isoform, are restricted to random patches in the interdigitation-free regions of the cortical fiber membrane. Neither the α1 nor α3 isoforms is observed, intact, on western blot analysis of membrane preparations from decapsulated bovine or human lenses. This would suggest degradation of these isoforms in the membrane of the fully differentiated cortical fiber. 

It is obvious that the regulation of monovalent cation homeostasis is rather complex in this syncytium, the lens. The importance of the active transport of ⁸⁶Rb⁺ by the fibercell Na,K-ATPase has already been established for the bovine lens in organ culture. ¹² The role of the polarized distribution of two distinct Na,K-ATPases in the membrane of the cells of the central epithelium remains to be defined. The importance of the subsequent degradation of the epithelial cell Na,K-ATPases in mature fibers also remains to be elucidated. Finally, the changes in monovalent cation homeostasis in humans with cataract may or may not be the result of Na,K-ATPase inhibition as currently believed. Changes in Na,K-ATPase distribution, synthesis, and degradation could be expected to play a role as well. 

 Supported by Grant EY07010 (MHG) from the National Eye Institute of the National Institutes of Health.

 Submitted for publication January 21, 1999; revised April 4, 1999; accepted April 28, 1999.

 Proprietary interest category: N.

 Corresponding author: Margaret H. Garner, Department of Anatomy and Cell Biology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107.

 

Figure 1.

 

Antibody specificity was determined by staining of western blots of SDS–PAGE separations of rat kidney (K), rat heart (H), and rat brain (B) microsomes with the affinity-purified antibodies (glycine fraction) to the IC and EC epitopes of the α1, α2, and α3 catalytic subunit isoforms of the Na,K-ATPase. SDS–PAGE separations were on 7% gels with 19 ± 2 mg of the appropriate microsomal fraction per lane. Primary antibody dilution was 1:10. Secondary antibody dilution was 1:1000. Between the IC and EC panels is a representative standard for reference; standard molecular weights are (from top to bottom), 133, 115 kda (faint), 79, and 49 kDa.

Figure 2.

 

Localization of Na,K-ATPase catalytic subunit isoforms of the bovine lens epithelium. Indirect immunofluorescence with the α1EC (A and E), α2EC (B and F), and α3EC (C and G) antisera and sections from the central epithelium (A through D) and sections from the equatorial epithelium with underlying superficial fibers (E through H). (D and H) Representative controls with normal rabbit serum and with the one of the TBST fractions (α2EC) from the affinity purification, respectively. Controls with the TBST fractions from the affinity purification of α1EC and α3EC were similar to (H), data not included.

Figure 3.

 

Localization of the Na,K-ATPase catalytic subunit isoforms of the cortex of decapsulated bovine (A, C, D, G) and human (B, E, F, H, I) lenses stained with α1EC (A through C), α2EC (D through F), or α3EC (G through I). (C, F, and I) Representative of stains with the TBST fractions from the affinity purification of α1EC,α 2EC, and α3EC, respectively. Magnification, ×100.

Figure 4.

 

Representative results for immunogold labeling of sections from the cortex of a human lens with α1EC (A), α2EC (B), and α3EC (C and D). (E) Normal rabbit serum control; (F) representative control with a TBST fraction from the affinity purification (α2EC). Magnification, ×50,000.

Figure 5.

 

Immunogold staining of membranes isolated from the bovine lens cortex with the affinity-purified α2IC antiserum (A), the affinity-purified α2EC antiserum (B), the TBST fraction from the affinity purification of α2IC (C), and the TBST fraction from the affinity purification of α2EC (D). Magnification, ×80,000.

Table 1.

 

Immunogold Labeling of Isolated Membrane Fractions

Antiserum	Bovine Renal Medulla* Microsomes			Human Lens Fiber Cell* Membranes			Bovine Lens Fiber Cell* Membranes
		TBST Fraction Particles/ 4 μm2	Gly Fraction Particles/ 4 μm2	TBST Fraction Particles/ 4 μm2	Gly Fraction Particles/ 4 μm2	TBST Fraction Particles/ 4 μm2	Gly Fraction Particles/ 4 μm2
α1IC	5 ± 3	21 ± 5, †	3 ± 3	4 ± 4	5 ± 4	5 ± 6
α1EC	5 ± 5	28 ± 10, †	4 ± 3	5 ± 3	5 ± 5	6 ± 4
α2IC	3 ± 3	5 ± 2	5 ± 2	77 ± 23, †	5 ± 3	23 ± 9, †
α2EC	4 ± 5	4 ± 1	5 ± 4	62 ± 30, †	5 ± 6	28 ± 10, †
α3IC	4 ± 3	6 ± 3	5 ± 4	28 ± 7, † , ‡	4 ± 5	5 ± 3
α3EC	2 ± 3	5 ± 3	4 ± 5	24 ± 10, † , ‡	4 ± 5	8 ± 5

 EC, extracellular; Gly, glycine; IC, intracellular; TBST, Tris buffer with NaCl and Tween.

*  Number of fields per antiserum fraction were 20, 35, and 48 for the renal medulla microsomes, human lens fiber cell membranes and bovine lens fibercell membranes, respectively.

†  Values significantly different between Gly and TBST fractions (the controls); P ≤ 0.001.

‡  Comparisons of results with glycine fractions α2IC to α3IC andα 2EC to α3EC in the human lens membrane preparations, P ≤ 0.001.

Figure 6.

 

Immunostaining of western blot analysis confirms the results of the immunocytochemistry studies. Membranes isolated from the cortex of decapsulated bovine lenses were separated by SDS–PAGE on 7% gels (lanes b through d, 30 μg protein/lane) with the prestained broad range SDS–PAGE standard (lane a) as a reference. Lane b is the result of staining of the gel with Coomassie blue; lane c is the result of staining of part of the western blot with α2IC; and lane d is the result of staining of portion of the western blot with α2EC. Microsomal fractions, isolated by differential centrifugation from the central epithelium and equatorial epithelium of bovine lenses, were separated by SDS–PAGE on 10% gels and transferred to nitrocellulose. Lanes e, h, and k show the results of staining of the sections of the western blot of the microsomal preparation of the central epithelium (20 μg protein/lane) with α1IC, α2IC, andα 3IC, respectively. Lanes f, i, and l show the results of staining of sections of the western blot of the microsomal preparation of the equatorial epithelium (15–20 μg protein/lane) with α1IC, α2IC, and α3IC, respectively. Lanes g, j, and m are the separated prestained low-range SDS–PAGE standard (Bio-Rad), included on each gel as a reference. A portion (50μ g protein) of the water insoluble fraction, isolated from a decapsulated human lens, was separated by SDS–PAGE on a 10% to 20% gradient gel and transferred to nitrocellulose. The blot was stained with the α2IC antiserum (lane n); the prestained high-range molecular-weight standard, run with the sample (lane o) as a reference.