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.