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The influence of cycloheximide on Na,K-ATPase activity in cultured human lens epithelial cells.
Invest Ophthalmol Vis Sci. 2002 Aug; 43(8):2714-20.IO

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 (alpha) 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 alpha subunit was determined by Western blot analysis. Synthesis of Na,K-ATPase alpha1 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 microM cycloheximide for 24 hours. However, as judged by Western blot density, the abundance of Na,K-ATPase alpha1 and alpha3 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 alpha1 subunit polypeptide within 30 minutes, consistent with alpha1 polypeptide synthesis. Na,K-ATPase alpha1 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 alpha1 subunit polypeptide of HLE-B3 cells. When added during the chase phase of the experiment, cycloheximide was found to slow the disappearance of labeled alpha1 polypeptide, consistent with a reduced rate of polypeptide degradation.

CONCLUSIONS

The results suggest that a continuous cycle of Na,K-ATPase alpha1 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.

Authors+Show Affiliations

Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

12147607

Citation

Cui, Guangming, et al. "The Influence of Cycloheximide On Na,K-ATPase Activity in Cultured Human Lens Epithelial Cells." Investigative Ophthalmology & Visual Science, vol. 43, no. 8, 2002, pp. 2714-20.
Cui G, Dean WL, Delamere NA. The influence of cycloheximide on Na,K-ATPase activity in cultured human lens epithelial cells. Invest Ophthalmol Vis Sci. 2002;43(8):2714-20.
Cui, G., Dean, W. L., & Delamere, N. A. (2002). The influence of cycloheximide on Na,K-ATPase activity in cultured human lens epithelial cells. Investigative Ophthalmology & Visual Science, 43(8), 2714-20.
Cui G, Dean WL, Delamere NA. The Influence of Cycloheximide On Na,K-ATPase Activity in Cultured Human Lens Epithelial Cells. Invest Ophthalmol Vis Sci. 2002;43(8):2714-20. PubMed PMID: 12147607.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The influence of cycloheximide on Na,K-ATPase activity in cultured human lens epithelial cells. AU - Cui,Guangming, AU - Dean,William L, AU - Delamere,Nicholas A, PY - 2002/7/31/pubmed PY - 2002/8/8/medline PY - 2002/7/31/entrez SP - 2714 EP - 20 JF - Investigative ophthalmology & visual science JO - Invest Ophthalmol Vis Sci VL - 43 IS - 8 N2 - PURPOSE: Earlier studies from this laboratory demonstrated the ability of lens epithelium to synthesize new Na,K-adenosine triphosphatase (Na,K-ATPase) catalytic subunit (alpha) 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 alpha subunit was determined by Western blot analysis. Synthesis of Na,K-ATPase alpha1 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 microM cycloheximide for 24 hours. However, as judged by Western blot density, the abundance of Na,K-ATPase alpha1 and alpha3 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 alpha1 subunit polypeptide within 30 minutes, consistent with alpha1 polypeptide synthesis. Na,K-ATPase alpha1 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 alpha1 subunit polypeptide of HLE-B3 cells. When added during the chase phase of the experiment, cycloheximide was found to slow the disappearance of labeled alpha1 polypeptide, consistent with a reduced rate of polypeptide degradation. CONCLUSIONS: The results suggest that a continuous cycle of Na,K-ATPase alpha1 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. SN - 0146-0404 UR - https://www.unboundmedicine.com/medline/citation/12147607/The_influence_of_cycloheximide_on_NaK_ATPase_activity_in_cultured_human_lens_epithelial_cells_ L2 - https://iovs.arvojournals.org/article.aspx?volume=43&issue=8&page=2714 DB - PRIME DP - Unbound Medicine ER -