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The Influence of Cycloheximide on Na,K-ATPase Activity in Cultured Human Lens Epithelial Cells

¹ From the Departments of Ophthalmology and Visual Sciences, ² Biochemistry and Molecular Biology, and ³ Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky.

PURPOSE. Earlier studies from this laboratory demonstrated the ability of lens epithelium to synthesize new Na,K-adenosine triphosphatase (Na,K-ATPase) catalytic subunit () polypeptide under conditions of increased ion permeability. In the present study, the authors considered whether continuous synthesis of Na,K-ATPase protein is necessary for maintenance of Na,K-ATPase activity in lens cells.

METHODS. Na,K-ATPase activity was measured by quantifying the ouabain-sensitive rate of ATP hydrolysis in cultured human lens epithelial cells (HLE-B3) permeabilized with digitonin. The abundance of Na,K-ATPase subunit was determined by Western blot analysis. Synthesis of Na,K-ATPase 1 polypeptide was investigated by measuring ³⁵S-methionine incorporation.

RESULTS. Na,K-ATPase activity was reduced to less than 20% of the control level in HLE-B3 cells exposed to 100 µM cycloheximide for 24 hours. However, as judged by Western blot density, the abundance of Na,K-ATPase 1 and 3 subunit in cycloheximide-treated cells was 90% and 84% of the control level, respectively. ³⁵S-methionine incorporation experiments revealed detectable labeling of Na,K-ATPase 1 subunit polypeptide within 30 minutes, consistent with 1 polypeptide synthesis. Na,K-ATPase 1 polypeptide labeling was also detected in the epithelium of intact rat lenses that had been allowed to incorporate ³⁵S-methionine. Cycloheximide abolished ³⁵S-methionine incorporation into Na,K-ATPase 1 subunit polypeptide of HLE-B3 cells. When added during the chase phase of the experiment, cycloheximide was found to slow the disappearance of labeled 1 polypeptide, consistent with a reduced rate of polypeptide degradation.

CONCLUSIONS. The results suggest that a continuous cycle of Na,K-ATPase 1 synthesis and degradation may occur in lens epithelial cells. Cycloheximide appeared to inhibit Na,K-ATPase protein synthesis and degradation. The observed reduction of Na,K-ATPase activity after treatment with cycloheximide indicates that even though Na,K-ATPase remains abundant, Na,K-ATPase becomes inactivated when protein synthesis is inhibited.

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