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

Abstract

In earlier studies we demonstrated that cyclical mechanical strain on vascular smooth muscle cells increases intracellular Na + and upregulates the α-1 and α-2 isoform expression of Na +,K +-ATPase, and that the increase of intracellular Na + and upregulation of the α-2 isoform expression are blocked by Gd 3+, 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 α 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 α-1 and α-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 μM 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 α isoform upregulation and the regulatory process requires less than one day.