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OBJECTIVE In a previous study, an increase in Na,K-ATPase alpha 2 expression was detected in the epithelium of porcine lenses exposed to amphotericin B, an ionophore that also increases lens sodium and stimulates active sodium transport. The purpose of the present study was to determine whether an increase of Na,K-ATPase alpha 2 synthesis is a response to an episode of rapid Na-K transport or whether the increase in lens sodium alone can initiate the response. METHODS Western blot analyses were conducted to probe for Na,K-ATPase alpha polypeptides in membrane material isolated from porcine lens epithelium. Ouabain-sensitive adenosine triphosphate hydrolysis was used as an index of Na,K-ATPase activity, and lens ion content was determined by atomic absorption spectrophotometry. 86-Rubidium (86Rb) uptake was measured as an indicator for active potassium transport. RESULTS 86Rb uptake was markedly diminished in lenses exposed to dihydro-ouabain (DHO), signifying inhibition of active sodium-potassium transport. Consistent with this, the sodium content of DHO-treated lenses increased. By western blot analysis, a marked increase of Na,K-ATPase alpha 2 polypeptide could be detected in the epithelium of DHO-treated lenses. To rule out the possibility that apparent stimulation of Na,K-ATPase alpha 2 synthesis stemmed from binding of DHO to Na,K-ATPase sites, experiments were conducted to confirm an increase of Na,K-ATPase alpha 2 polypeptide in the epithelium of lenses exposed to low-potassium medium to inhibit active sodium-potassium transport. Consistent with the apparent increase of Na,K-ATPase polypeptide, Na,K-ATPase activity was detectably increased in epithelial material isolated from lenses pretreated with DHO or low-potassium medium. CONCLUSIONS An increase in Na,K-ATPase alpha 2 polypeptide can occur in the epithelium of lenses subjected to an episode of sodium pump inhibition. This suggests the response could be triggered by an increase in cell sodium and does not necessarily require a period of stimulated active sodium-potassium transport.
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