The influence of cycloheximide on Na,K-ATPase activity in cultured human lens epithelial cells

Guangming Cui, William L. Dean, Nicholas A. Delamere

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

PURPOSE. Earlier studies from this laboratory demonstrated the ability of lens epithelium to synthesize new Na,K-adenosine triphosphatase (Na,K-ATPase) catalytic subunit (α) 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 α subunit was determined by Western blot analysis. Synthesis of Na,K-ATPase α1 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 α1 and α3 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 α1 subunit polypeptide within 30 minutes, consistent with α1 polypeptide synthesis. Na,K-ATPase α1 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 α1 subunit polypeptide of HLE-B3 cells. When added during the chase phase of the experiment, cycloheximide was found to slow the disappearance of labeled α1 polypeptide, consistent with a reduced rate of polypeptide degradation. CONCLUSIONS. The results suggest that a continuous cycle of Na,K-ATPase α1 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.

Original languageEnglish (US)
Pages (from-to)2714-2720
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume43
Issue number8
StatePublished - Aug 5 2002
Externally publishedYes

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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