Effect of Na+ on Na+,K+-ATPase alpha-subunit expression and Na+-pump activity in aortic smooth muscle cells

In earlier studies we demonstrated that cyclical mechanical strain on vascular smooth muscle cells increases intracellular Na+ and upregulates the alpha-1 and alpha-2 isoform expression of Na+,K+-ATPase, and that the increase of intracellular Na+ and upregulation of the alpha-2 isoform expression are blocked by Gd3+, which blocks entry of ions (including Na+) through stretch-activated channels. The present study was designed to investigate the role of intracellular Na+ in Na+,K+-ATPase regulation by increasing intracellular Na+ with chronic ouabain treatment. In parallel experiments, we measured Na+,K+-ATPase alpha isoform expression, Na+-pump activity and intracellular Na+ in cultured aortic smooth muscle cells after treatment with two concentrations of ouabain for various time periods. Treatment with 100 nM ouabain resulted in a significant elevation in intracellular Na+ after 1 (21%) and 2 h (12%), but the value returned to baseline after 12 h. Both alpha-1 and alpha-2 subunits of Na+,K+-ATPase were significantly upregulated after 1 through 4 days. Na+-pump activity was also stimulated, and the time course of this effect closely followed protein expression. At 200 microM of ouabain, the effects on intracellular Na+, isoform expression and Na+-pump activity at earlier time points (1 h through 1 day) were similar to those with 100 nM treatment, but prolonged treatment (2 and 4 days) resulted in an accumulation of intracellular Na+ and inhibition of the isoform expression and Na+-pump activity, possibly due to general dysfunction of the cells as a result of chronic exposure to high concentrations of ouabain. We conclude that elevated intracellular Na+ can serve as a signal to mediate the alpha isoform upregulation and the regulatory process requires less than one day.