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 alpha1 and alpha3 catalytic subunit isoforms in central epithelium. In the cortical fibers, the alpha2 isoform is present around the interdigitations. The alpha3 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.