NA,K-ATPASE RESPONSE TO OSMOTIC-STRESS IN PRIMARY DOG LENS EPITHELIAL-CELLS

Purpose, Na,K-ATPase activity increases in lens cells exposed to hyper tonic stress. To test whether the increase in activity involves stimul ation of Na,K-ATPase expression, dog lens epithelial cells were subjec ted to hypertonic stress, and the time course of Na,K-ATPase protein a nd mRNA response was measured. Methods. Primary cultures of dog lens e pithelial cells were maintained in isotonic or hypertonic media over t he course of several days. Rubidium-86 uptake measurements, immunoreac tive protein, and northern blot analysis were performed. Results, Dog lens epithelial cells exposed to hypertonic stress from culture medium supplemented with 150 mM NaCl or 250 mM cellobiose showed a twofold i ncrease in Na,K-ATPase activity. The increase in activity was blocked by cycloheximide and was reversible when the cells were returned to is otonic medium. This activity was unaffected by the aldose reductase in hibitor, tolrestat. Na,K-ATPase protein and mRNA levels increased in c ells exposed to medium containing 150 mM NaCl. Northern blot analysis showed that the alpha-1, and beta-1 mRNA levels increased as early as 6 hours and maximally increased 1.5-fold to twofold by 12 to 24 hours. Conclusions. Elevation of Na,K-ATPase activity in dog lens epithelial cells exposed to hypertonic stress was associated with increased expr ession of Na,K-ATPase subunit mRNAs and was dependent on protein synth esis. These results suggest that upregulation of the enzyme activity i s the result of an induction of Na,K-ATPase.