The influence of cycloheximide on Na,K-ATPase activity in cultured human lens epithelial cells.
OBJECTIVE
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.
Cui
Guangming
G
Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA.
Dean
William L
WL
Delamere
Nicholas A
NA
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology*; D008715:Methionine / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D011500:Protein Synthesis Inhibitors; D003513:Cycloheximide; D008715:Methionine; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium