May 2002
Volume 43, Issue 5
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Physiology and Pharmacology  |   May 2002
Altered Na,K-ATPase Pattern in γ-Crystallin Mutant Mice
Author Affiliations
  • Amy Moseley
    From the Department of Ophthalmology and Visual Sciences, University of Louisville, Louisville, Kentucky; the
    Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio; and
  • Jochen Graw
    GSF-National Research Center for Environment and Health, Institute of Mammalian Genetics, Neuherberg, Germany.
  • Nicholas A. Delamere
    From the Department of Ophthalmology and Visual Sciences, University of Louisville, Louisville, Kentucky; the
Investigative Ophthalmology & Visual Science May 2002, Vol.43, 1517-1519. doi:
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    • Get Citation

      Amy Moseley, Jochen Graw, Nicholas A. Delamere; Altered Na,K-ATPase Pattern in γ-Crystallin Mutant Mice. Invest. Ophthalmol. Vis. Sci. 2002;43(5):1517-1519.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Na,K-adenosine triphosphatase (ATPase) activity is elevated in the lenses of murine cataract Cryge t and Cryge ns mutant mice. In the present study, the expression of Na,K-ATPase α1, α2, and α3 catalytic subunit polypeptides was examined in the lenses of these mutant mice.

methods. Membrane material was isolated from lenses and brain of 3-week-old wild-type mice, as well as heterozygous and homozygous mutant mice. Microsomal membranes were prepared by centrifugation of the homogenized material, and Na,K-ATPase polypeptides were detected by immunoblot analysis with antibodies directed against the Na,K-ATPase isoforms α1, α2, and α3.

results. For the Na,K-ATPase isoforms α2 and α3, membrane material obtained from the homozygous cataract mutants showed dense immunoblot bands that were not detected in material obtained from wild-type mice. An apparent increase of the α1 Na,K-ATPase isoform band density was also detected in lens material from the homozygous mutant mice. The Na,K-ATPase α3 polypeptide was also detected in lens membrane material obtained from heterozygous mice of both mutant strains. The α2 Na,K-ATPase polypeptide was observed in lens membrane material obtained from heterozygous Cryge t mice, and a less dense band was detected in heterozygous Cryge ns mice. Band densities of Na,K-ATPase subunits α1, α2, and α3 detected in brain membrane material were similar in both mutant and wild-type mice.

conclusions. The immunoblot results suggest that the abundance of Na,K-ATPase polypeptide is increased in the lens of the cataract mouse mutant but is not altered in the brain. The expression of the α2 and α3 isoform proteins of Na,K-ATPase is markedly upregulated in the cataractous lens.

Cytoplasmic sodium and potassium concentrations within the lens are maintained by Na,K-adenosine triphosphatase (ATPase), which actively extrudes sodium and imports potassium. Because many cataractous lenses display an increased level of sodium, 1 there has been interest in the possibility that changes in this active sodium-potassium transport mechanism may be associated with cataract. In studies of murine cataract mutant mice, one of the unanticipated biochemical alterations in the lens was an increase of Na,K-ATPase activity. 2 3 4 In the present study we examined whether the abundance of Na,K-ATPase protein is altered in lenses from these mutant mice. 
Cryge t and Cryge ns mutant mice have strong linkage to the Cryg gene cluster encoding γ-crystallin 5 and have each been characterized as mutations within distinct Cryg genes. 6 The cataract mutant, Cryge t (previous gene symbols, R-324; Cat2 t ), was detected among offspring after paternal radiation exposure 7 and described as having total, cloudy lens opacity. Other ocular changes included microphthalmia with small pupils and iris dysplasia. Histologic examination revealed a complex disruption of the cellular organization of the lens. 4 A point mutation in the third exon of Cryge t was found that predicts a truncation of the protein after amino acid 143 of the γE-crystallin. 6  
A second mutation referred to as Cryge ns (previous gene symbols: K-134; Scat) was demonstrated to be allelic to the other Cryg mutants 8 and is characterized by a deletion in the third exon of Cryge t affecting more than 2 kb of the γE-crystallin gene. 9 This causes an opacity at the anterior suture of the lens in heterozygotes and microphthalmia with vacuolated lenses in homozygotes. Heterozygotes exhibit hydropic swelling of lens epithelium, whereas in homozygotes there is marked interruption and degeneration of lens fibers as well as clefts and folds in the lens capsule. 3  
Heterozygous and homozygous Cryge ns and Cryge t mutant mice have been partially characterized by biochemical alterations that occur in the juvenile lens. A decrease in water-soluble lens proteins correlates with the smaller size of the cataractous lenses. The cataractous lenses exhibit slightly higher concentrations of glucose and glucose-6-phosphate and the concentration of oxidized glutathione (GSSG), an indicator of oxidative stress, is increased two- to fourfold. There is also an increase of lens water content. However, one of the more distinct abnormalities in the mutant mice is a 2.5- to 6.5-fold increase in Na,K-ATPase activity in the juvenile lenses. 2 3 4 The mutant mice have a decreased lens adenosine triphosphate (ATP) content that could be due in part to the high Na,K-ATPase activity’s causing depletion of ATP. 
Na,K-ATPase is composed of a catalytic α subunit and a glycoprotein β subunit. Multiple isoforms of both subunits have been identified. The α1 and β1 isoforms are found in all cell types but only certain tissues express the α2, α3, α4, β2, and β3 isoforms. 10 11 12 13 Lens cells appear capable of effecting significant changes in Na,K-ATPase protein expression. In cultured porcine lenses exposed to either amphotericin B or dihydro-ouabain, treatments that cause cytoplasmic sodium to increase, a significant increase in Na,K-ATPase activity in the lens epithelium was associated with an increase in Na,K-ATPase α2 isoform polypeptide levels. 14 15 16 Because the cataract mutants Cryge ns and Cryge t exhibit increased Na,K-ATPase activity, the present study was performed to test whether the increase in activity may be associated with a change in the abundance of specific Na,K-ATPase polypeptides. 
Methods
Membrane material was isolated from lenses of 3-week-old mice. The animal studies were performed in the GSF-National Research Center for Environment and Health animal facility according to the German Law on the Protection of Animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Samples were obtained from wild-type mice or heterozygous or homozygous Cryge ns or Cryge t mutant animals. Western blot experiments were conducted with antibodies directed against the α1, α2, and α3 isoforms of Na,K-ATPase (see Moseley et al. 17 for details of the method used). The antibodies used were α6f, specific for the α1 Na,K-ATPase isoform (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa), 18 McB2, specific for the α2 Na,K-ATPase isoform (kindly provided by Kathy Sweadner; Harvard University) 19 and MA3-915, specific for the α3 Na,K-ATPase isoform (Affinity Bioreagents, Golden, CO). 
Results
The results are shown in Figure 1 . For Na,K-ATPase α2 and α3 isoforms, lens membrane material samples obtained from the homozygous mutants exhibited dense immunoblot bands at 100 kDa that were not detectable in wild-type lens samples. Membrane material from heterozygous mutants also showed a dense Na,K-ATPase α3 band. A dense Na,K-ATPase α2 band was observed in lens samples from the Cryge t but not the Cryge ns mutant, in which only a faint band was detected. A detectable increase in band density of the Na,K-ATPase isoform α1 compared with the wild type was detected in lens membrane material obtained from homozygous and heterozygous Cryge t mutants and the homozygous Cryge ns mutant. 
Although detectable changes of Na,K-ATPase immunoreactive polypeptide abundance were observed in lens tissue from the Cryge ns and Cryge t mice, no apparent change in α1, α2, or α3 Western blot band density was observed in membrane material isolated from brain of the mutant mice (Fig. 1B) . This supports the notion that changes in Na,K-ATPase expression resulting from the γE-crystallin mutations are confined to the lens and are not a general tissue phenomenon. The detection of the three different Na,K-ATPase α-subunit isoforms in brain is consistent with previous studies of Na,K-ATPase distribution in different tissues. 10 Isoform selectivity of the Na,K-ATPase α2 and α3 isoform antibodies used in this study was confirmed by Western blot analysis, showing detection of α2 in heart, skeletal muscle, and brain, but not kidney, and the detection of α3 in brain, but not skeletal muscle, heart, or kidney. 
Discussion
The immunoblot results suggest there is an increase of Na,K-ATPase polypeptide abundance in the lens of Cryge ns and Cryge t mutants, particularly the abundance of α2 and α3 isoforms that do not appear at detectable levels in the wild-type mouse lens. This finding is consistent with the previously reported increase of lens Na,K-ATPase activity in these two mouse mutants. The increased water content that is found in lenses of the cataract mutants suggests the lenses may have increased membrane permeability. It is possible that such an increase causes lens cells to upregulate expression of the Na,K-ATPase α2 and α3 isoforms. A link between increased lens membrane permeability, increased cytoplasmic sodium, and an increase in expression of Na,K-ATPase α2 has been proposed earlier, and we suggested that α2 could be an inducible isoform of Na,K-ATPase in porcine lens. 14 15 16 To our knowledge this is the first time lens cells have been shown to increase the abundance of Na,K-ATPase α3 protein. It remains to be determined whether expression of Na,K-ATPase α2 and α3 serves a special function that would not be performed if the lens responded by simply increasing the abundance of Na,K-ATPase α1, the housekeeping Na,K-ATPase isoform. 
Figure 1.
 
(A) Na,K-ATPase isoforms were detectable in the lens membranes obtained from two different γ-crystallin-deficient mutant mice. Lens membrane material was prepared from 3-week-old wild-type (WT) mice and heterozygous (+/−) or homozygous (−/−) Cryge ns and Cryge t mutant mice. The entire lens was used in each case, and lenses were pooled. The small size of the lenses, coupled with likely differences between wild-type and cataractous lenses in the efficiency of detachment of the capsule-epithelium from the fiber mass, did not permit separation of epithelium and fiber cells. Membrane protein (50 μg) was applied to each lane. The membrane proteins were separated by electrophoresis on a 7.5% SDS-polyacrylamide gel and blotted onto a polyvinylidene difluoride (PVDF) membrane. Western blot analysis for three different Na,K-ATPase isoforms (α1, α2, and α3) was performed as described previously. 18 Molecular weight markers were used in the range 115 to 38 kDa. The Na,K-ATPase α subunit appeared as a band at approximately 100 kDa. (B) Na,K-ATPase isoforms detected in membrane material isolated from brain of wild-type and mutant mice. To confirm antibody specificity in mouse tissues, Western blot analyses (C) for Na,K-ATPase α1, α2, and α3 were conducted using membrane material isolated from brain (Br), skeletal muscle (Sm), heart (Ht), and kidney (K) of adult wild-type mice. Details and methods used for membrane preparation are published. 18
Figure 1.
 
(A) Na,K-ATPase isoforms were detectable in the lens membranes obtained from two different γ-crystallin-deficient mutant mice. Lens membrane material was prepared from 3-week-old wild-type (WT) mice and heterozygous (+/−) or homozygous (−/−) Cryge ns and Cryge t mutant mice. The entire lens was used in each case, and lenses were pooled. The small size of the lenses, coupled with likely differences between wild-type and cataractous lenses in the efficiency of detachment of the capsule-epithelium from the fiber mass, did not permit separation of epithelium and fiber cells. Membrane protein (50 μg) was applied to each lane. The membrane proteins were separated by electrophoresis on a 7.5% SDS-polyacrylamide gel and blotted onto a polyvinylidene difluoride (PVDF) membrane. Western blot analysis for three different Na,K-ATPase isoforms (α1, α2, and α3) was performed as described previously. 18 Molecular weight markers were used in the range 115 to 38 kDa. The Na,K-ATPase α subunit appeared as a band at approximately 100 kDa. (B) Na,K-ATPase isoforms detected in membrane material isolated from brain of wild-type and mutant mice. To confirm antibody specificity in mouse tissues, Western blot analyses (C) for Na,K-ATPase α1, α2, and α3 were conducted using membrane material isolated from brain (Br), skeletal muscle (Sm), heart (Ht), and kidney (K) of adult wild-type mice. Details and methods used for membrane preparation are published. 18
 
The authors thank Erika Bürkle for the expert technical assistance. 
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